1194 Citations
Various techniques have been used to detect crystallization in amorphous solid dispersions ASD However most of these techniques do not enable the detection of very low levels of crystallinity The aim ofthe current study was to compare the sensitivity of second harmonic generation SHG microscopy with powder X-ray diffraction XRPD in detecting the presence of crystals in low drug loading amorphous solid dispersions Amorphous solid dispersions of the poorly water soluble compounds flutamide FTM wt drug loading and ezetimibe EZT wt drug loading with hydroxypropyl methylcellulose acetate succinate HPMCAS were prepared by spray drying To induce crystallization samples were subsequently ... More
Various techniques have been used to detect crystallization in amorphous solid dispersions (ASD). However, most of these techniques do not enable the detection of very low levels of crystallinity (<1%). The
aim ofthe current study was to compare the sensitivity of second harmonic generation (SHG) microscopy
with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble compounds, flutamide
(FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To induce crystallization, samples
were subsequently stored at 75% or 82% relative humidity (RH) and 40 ?C. Crystallization was monitored
by XRPD and by SHG microscopy. Solid state nuclear magnetic resonance spectroscopy (ssNMR) was
used to further investigate crystallinity in selected samples. For flutamide, crystals were detected by
SHG microscopy after 8 days of storage at 40 ?C/82% RH, whereas no evidence of crystallinity could be
observed by XRPD until 26 days. Correspondingly, for FTM samples stored at 40 ?C/75% RH, crystals were
detected after 11 days by SHG microscopy and after 53 days by XRPD. The evolution of crystals, that is
an increase in the number and size of crystalline regions, with time could be readily monitored from the
SHG images, and revealed the formation of needle-shaped crystals. Further investigation with scanning
electron microscopy indicated an unexpected mechanism of crystallization, whereby flutamide crystals
grew as needle-shaped projections from the surface of the spray dried particles. Similarly, EZT crystals
could be detected at earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days).
Thus, SHG microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of crystals formed from spray dried particles, providing much earlier detection of crystallinity than
XRPD under comparable run times. Less
aim ofthe current study was to compare the sensitivity of second harmonic generation (SHG) microscopy
with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble compounds, flutamide
(FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To induce crystallization, samples
were subsequently stored at 75% or 82% relative humidity (RH) and 40 ?C. Crystallization was monitored
by XRPD and by SHG microscopy. Solid state nuclear magnetic resonance spectroscopy (ssNMR) was
used to further investigate crystallinity in selected samples. For flutamide, crystals were detected by
SHG microscopy after 8 days of storage at 40 ?C/82% RH, whereas no evidence of crystallinity could be
observed by XRPD until 26 days. Correspondingly, for FTM samples stored at 40 ?C/75% RH, crystals were
detected after 11 days by SHG microscopy and after 53 days by XRPD. The evolution of crystals, that is
an increase in the number and size of crystalline regions, with time could be readily monitored from the
SHG images, and revealed the formation of needle-shaped crystals. Further investigation with scanning
electron microscopy indicated an unexpected mechanism of crystallization, whereby flutamide crystals
grew as needle-shaped projections from the surface of the spray dried particles. Similarly, EZT crystals
could be detected at earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days).
Thus, SHG microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of crystals formed from spray dried particles, providing much earlier detection of crystallinity than
XRPD under comparable run times. Less
Pseudomonas aeruginosa a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis uses three interconnected quorum-sensing systems to coordinate virulence processes At variance with other Gram-negative bacteria one of these systems relies on -alkyl- H -quinolones Pseudomonas quinolone signal PQS and might hence be an attractive target for new anti-infective agents Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA the first enzyme of PQS biosynthesis in complex with anthraniloyl-AMP and with -fluoroanthraniloyl-AMP FABA-AMP at and resolution We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino ... More
Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7 Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P. aeruginosa infections. Less
NADH and NADPH are redox coenzymes broadly required for energy metabolism biosynthesis and detoxification Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking in part because of a lack of tools with which to investigate them in living cells We previously reported the use of the naturally occurring Lactobacillus brevis H O-forming NADH oxidase LbNOX as a genetic tool for manipulation of the NAD NADH ratio in human cells Here we present TPNOX triphosphopyridine nucleotide oxidase a rationally designed and engineered mutant ... More
NADH and NADPH are redox coenzymes broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking, in part because of a lack of tools with which to investigate them in living cells. We previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here we present TPNOX (triphosphopyridine nucleotide oxidase), a rationally designed and engineered mutant of LbNOX that is strictly specific towards NADPH. We characterize the effects of TPNOX expression on cellular metabolism and use it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected. Less
DHHC enzymes catalyze palmitoylation a major post-translational modification that regulates a number of key cellular processes There are up to DHHCs in mammals and hundreds of substrate proteins that get palmitoylated However how DHHC enzymes engage with their substrates is still poorly understood There is currently no structural information about the interaction between any DHHC enzyme and protein substrates In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC ANK and Snap b We solved a high-resolution crystal structure of the complex between ANK and a peptide fragment of ... More
DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most relevant substrates. Less
The growing demand for fast highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device allowing the assessment of critical steps and consequently providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum by identifying the HRP biomarker The best device performance was ... More
The growing demand for fast, highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field. This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms. The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device, allowing the assessment of critical steps and, consequently, providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum, by identifying the HRP2 biomarker. The best device performance was achieved by using 0,05 µg (1 µL / 50 µg mL-1) of the capture antibody and incubation for 5 minutes for its adsorption; blocking of nitrocellulose with 1.5% BSA (m/v) containing 0.1% of the surfactant Tween-20 (v/v) by immersion and incubation for 10 minutes; 0.04 µg (20 µL/2 µg mL-1) detection antibody conjugated to the enzyme peroxidase; washing with 0.01 mol L-1 Tris-HCl buffer solution pH 7.4 containing 0.15 mol L-1 NaCl and 0.1% Tween 20 (v/v); addition of 5 µL of TMB chromogenic substrate and reading within 5-20 minutes after this addition. The colorimetric detection system had a visual detection limit of 5 ng mL-1 (135 pmol L-1), which value should be sufficient to identify malaria contamination on the first day of symptom onset.The developed platform was then applied to blood samples from patients infected by the disease, demonstrating efficiency in the qualitative discrimination of a positive and negative result and the generation of reliable results. In another study, with the aim of providing a second generation of the device, a paper-based 3D detection system capable of being coupled to a lateral flow immunoassay platform was built. This system enables quantitative, automated diagnosis with signal amplification through the incorporation of a new polymeric material of the class of poly(benzyl ethers) that selectively responds to hydrogen peroxide, in addition to requiring only color and color visualization. a timer for obtaining data and later analyzing the results.The delimitation of hydrophobic barriers on the paper was carried out by means of wax printing and the orientation processes and type of paper to be used in the layer containing the polymer were evaluated. The material deposition process on the paper surface demonstrated greater repeatability when performed automatically by means of a liquid handling robot. The highest sensitivity condition for the 3D detection system was achieved using a polymer concentration of 4.0 mg mL-1 with the achievement of a detection limit of 0.02 mmol L-1 of hydrogen peroxide. This system was then coupled to a lateral flow immunoassay platform and used in initial studies to detect the creatine kinase MB isoenzyme, one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU)one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU) Less
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases Given the importance of G protein-coupled receptors GPCRs as pharmaceutical targets there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs Here we present the - resolution structure of a complex between the human -hydroxytryptamine B -HT B receptor and an antibody Fab fragment bound to the extracellular side of the receptor determined by serial femtosecond crystallography with an X-ray free-electron laser The antibody binds to a D epitope of the receptor that includes all three extracellular loops The -HT ... More
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. Less
Vancomycin is known to bind to Zn II and can induce a zinc starvation response in bacteria Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn II crystals and fibre X-ray diffraction Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala Polymerization occurs via interaction of Zn II with the N-terminal methylleucine group of vancomycin and we show that the activity of other glycopeptide antibiotics with this ... More
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors. Less
The cannabinoid receptor CB is the principal target of the psychoactive constituent of marijuana the partial agonist -tetrahydrocannabinol -THC Here we report two agonist-bound crystal structures of human CB in complex with a tetrahydrocannabinol AM and a hexahydrocannabinol AM at and resolution respectively The two CB agonist complexes reveal important conformational changes in the overall structure relative to the antagonist-bound state including a reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region In addition a twin toggle switch of Phe and Trp superscripts denote Ballesteros Weinstein numbering is experimentally observed ... More
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties. Less
Lipoproteins serve essential roles in the bacterial cell envelope The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes All are potential targets for the development of new antibiotics Here we report the crystal structure of the last enzyme in the pathway apolipoprotein N-acyltransferase Lnt responsible for adding a third acyl chain to the lipoprotein s invariant diacylated N-terminal cysteine Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved they are remarkably similar Both consist of a membrane domain on which sits a globular periplasmic domain The active site resides above the membrane interface where the ... More
Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein�s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects. Less
High protein titers are gaining importance in biopharmaceutical industry A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity The complexity of the molecule itself as well as the various molecular interactions make it difficult to describe their solution behavior To study the formulation stability long- and short-range interactions and the formation of complex network structures have to be taken into account For a better understanding of highly concentrated solutions we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb ... More
High protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50–200 mg/ml at pH 5–9 with and without glycine, PEG4000, and Na2SO4 were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability. Less
The androgen receptor AR NR C is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation Pathological processes involving AR include Kennedy s disease and Klinefelter s syndrome as well as prostate ovarian and testicular cancer Strict regulation of sex hormone signaling is required for normal reproductive organ development and function Therefore testing small molecules for their ability to modulate AR is a first step in ... More
The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy’s disease and Klinefelter’s syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR β-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation. Less
The human glucagon receptor GCGR belongs to the class B G protein-coupled receptor GPCR family and plays a key role in glucose homeostasis and the pathophysiology of type diabetes Here we report the crystal structure of full-length GCGR containing both extracellular domain ECD and transmembrane domain TMD in an inactive conformation The two domains are connected by a -residue segment termed the stalk which adopts a -strand conformation instead of forming an -helix as observed in the previously solved structure of GCGR-TMD The first extracellular loop ECL exhibits a -hairpin conformation and interacts with the stalk to form a compact ... More
The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs. Less
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis the most common chronic inflammatory disease in humans worldwide Its comorbidity with systemic diseases such as type diabetes oral cancers and cardiovascular diseases continues to generate considerable interest Surprisingly these two microorganisms do not ferment carbohydrates rather they use proteinaceous substrates as carbon and energy sources However the underlying biochemical mechanisms of their energy metabolism remain unknown Here we show that dipeptidyl peptidase DPP a central metabolic enzyme in these bacteria undergoes a conformational change upon peptide binding to distinguish substrates from end products It binds substrates through an ... More
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme (“entropy reservoirs”). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors. Less
Apelin receptor APJR is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands apelin and Elabela each with different isoforms diversified by length and amino acid sequence Here we report the - resolution crystal structure of human APJR in complex with a designed -amino-acid apelin mimetic peptide agonist The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode Combined with mutation analysis and molecular dynamics simulations with apelin- binding to the wild-type APJR this structure provides a mechanistic understanding of apelin recognition and binding specificity Comparison of ... More
Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-� resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism. Less
Bacteria and archaea use the CRISPR Cas system as an adaptive response against infection by foreign nucleic acids Owing to its remarkable flexibility this mechanism has been harnessed and adopted as a powerful tool for genome editing The CRISPR Cas system includes two classes that are subdivided into six types and subtypes according to conservation of the cas gene and loci organization Recently a new protein with endonuclease activity belonging to class type V has been identified This endonuclease termed Cpf in complex with a single CRISPR RNA crRNA is able to recognize and cleave a target DNA preceded by ... More
Bacteria and archaea use the CRISPR�Cas system as an adaptive response
against infection by foreign nucleic acids. Owing to its remarkable flexibility, this
mechanism has been harnessed and adopted as a powerful tool for genome
editing. The CRISPR�Cas system includes two classes that are subdivided into
six types and 19 subtypes according to conservation of the cas gene and loci
organization. Recently, a new protein with endonuclease activity belonging to
class 2 type V has been identified. This endonuclease, termed Cpf1, in complex
with a single CRISPR RNA (crRNA) is able to recognize and cleave a target
DNA preceded by a 50
-TTN-30 protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of
Cpf1, the crystallization of an active complex containing the full extent of the
crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding
Cpf1 from Francisella novicida was cloned, overexpressed and purified.
The crRNA was transcribed and purified in vitro. Finally, the ternary
FnCpf1�crRNA�DNA complex was assembled and purified by preparative
electrophoresis before crystallization. Crystals belonging to space group C2221,
with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 A� , were obtained and
subjected to preliminary diffraction experiments. Less
against infection by foreign nucleic acids. Owing to its remarkable flexibility, this
mechanism has been harnessed and adopted as a powerful tool for genome
editing. The CRISPR�Cas system includes two classes that are subdivided into
six types and 19 subtypes according to conservation of the cas gene and loci
organization. Recently, a new protein with endonuclease activity belonging to
class 2 type V has been identified. This endonuclease, termed Cpf1, in complex
with a single CRISPR RNA (crRNA) is able to recognize and cleave a target
DNA preceded by a 50
-TTN-30 protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of
Cpf1, the crystallization of an active complex containing the full extent of the
crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding
Cpf1 from Francisella novicida was cloned, overexpressed and purified.
The crRNA was transcribed and purified in vitro. Finally, the ternary
FnCpf1�crRNA�DNA complex was assembled and purified by preparative
electrophoresis before crystallization. Crystals belonging to space group C2221,
with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 A� , were obtained and
subjected to preliminary diffraction experiments. Less
Clinical studies indicate that partial agonists of the G-protein-coupled free fatty acid receptor GPR enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type diabetes mellitus Full allosteric agonists AgoPAMs of GPR bind to a site distinct from partial agonists and can provide additional efficacy We report the - crystal structure of human GPR hGPR in complex with both the partial agonist MK- and an AgoPAM which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle Comparison with an additional - structure of the hGPR MK- binary complex reveals an induced-fit conformational coupling between ... More
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands. Less
Antibiotic-resistant bacterial infections are increasingly prevalent worldwide and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms One potential antibiotic target is the bacterial single-stranded DNA binding protein SSB which serves as a hub for DNA repair recombination and replication Eight highly conserved residues at the C-terminus of SSB use direct protein protein interactions PPIs to recruit more than a dozen important genome maintenance proteins to single-stranded DNA Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial ... More
Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy. Less
Schistosoma mansoni is the parasite responsible for schistosomiasis a disease that affects about million people worldwide Currently both direct treatment and disease control initiatives rely on chemotherapy using a single drug praziquantel Concerns over the possibility of resistance developing to praziquantel have stimulated efforts to develop new drugs for the treatment of schistosomiasis Schistosomes do not have the de novo purine biosynthetic pathway and instead depend entirely on the purine salvage pathway to supply its need for purines The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis Adenylosuccinate lyase SmADSL is an ... More
Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5′-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway. Less
Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets Computational as well as biophysical approaches based on nuclear magnetic resonance NMR fragment screening are powerful approaches but suffer from specific constraints that limit their usage Here we demonstrate the applicability of high-throughput thermal scanning HTTS as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening HTS The few ... More
Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets. Computational as well as biophysical approaches based on nuclear magnetic resonance (NMR) fragment screening are powerful approaches but suffer from specific constraints that limit their usage. Here, we demonstrate the applicability of high-throughput thermal scanning (HTTS) as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening. By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening (HTS). The few divergences for targets of low ligandability originate from the sensitivity differences of the orthogonal biophysical methods. We thus applied a new strategy making use of modulations in the solvent structure to improve assay sensitivity. This novel approach enables improved ligandability assessments in accordance with NMR-based assessments and more importantly positions the methodology as a valuable option for biophysical fragment screening. Less
PP C phosphatases control biological processes including stress responses development and cell division in all kingdoms of life Diverse regulatory domains adapt PP C phosphatases to specific functions but how these domains control phosphatase activity was unknown We present structures representing active and inactive states of the PP C phosphatase SpoIIE from Bacillus subtilis Based on structural analyses and genetic and biochemical experiments we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor Our analysis indicates that this switch is widely conserved among PP C family members serving as a platform ... More
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. Less
The Smoothened receptor SMO belongs to the Class Frizzled of the G protein-coupled receptor GPCR superfamily constituting a key component of the Hedgehog signalling pathway Here we report the crystal structure of the multi-domain human SMO bound and stabilized by a designed tool ligand TC using an X-ray free-electron laser source at The structure reveals a precise arrangement of three distinct domains a seven-transmembrane helices domain TMD a hinge domain HD and an intact extracellular cysteine-rich domain CRD This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation By combining the structural data ... More
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs. Less
The glucagon-like peptide- receptor GLP- R and the glucagon receptor GCGR are members of the secretin-like class B family of G-protein-coupled receptors GPCRs and have opposing physiological roles in insulin release and glucose homeostasis The treatment of type diabetes requires positive modulation of GLP- R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner Here we report crystal structures of the human GLP- R transmembrane domain in complex with two different negative allosteric modulators PF- and NNC at and resolution respectively The structures reveal a common binding pocket for negative allosteric modulators present in both GLP- R ... More
The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis1. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner2. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs4,5,6. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling. Less
We describe a fast easy and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide single-wavelength anomalous diffraction I-SAD The potential universality of the method is based on a common feature of membrane proteins the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins including a human G protein coupled receptor GPCR and we show that I-SAD can be applied using data collection strategies based on either standard or ... More
We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide–single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins—the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein–coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques. Less
Lipidic cubic phase LCP has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment Application of this material to a wide variety of membrane proteins however is hindered due to a limited number of available host lipids mostly monoacylglycerols MAGs Here we designed synthesized and characterized a series of chemically stable lipids resistant to hydrolysis with properties complementary to the widely used MAGs In order to assess their potential to serve as host lipids for crystallization we characterized the phase properties and lattice parameters of mesophases made of ... More
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C. Less
Neisserial heparin-binding antigen NHBA is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero the crystal structures of two fragment antigen-binding domains Fabs isolated from human monoclonal antibodies targeting NHBA were determined Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs these structures provide broad insights into the NHBA epitopes recognized by the human immune system As expected these Fabs also show remarkable structural conservation ... More
Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA. Less
Second harmonic generation SHG was integrated with Raman spectroscopy for the analysis of pharmaceutical materials Particulate formulations of clopidogrel bisulphate were prepared in two crystal forms Form I and Form II Image analysis approaches enable automated identification of particles by bright field imaging followed by classification by SHG Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with confidence in a total measurement time of ms per particle Complementary measurements by Raman and synchrotron XRD are in excellent agreement with the classifications made by SHG with measurement times of minute and several seconds per particle respectively ... More
Second harmonic generation (SHG) was integrated with Raman spectroscopy for the
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper The effect of trehalose and SU- epoxy novolac resin SU- on the stability of horseradish peroxidase HRP was studied in both a short-term experiment where the activity of various concentrations of HRP dried on paper were measured after h and a long-term experiment where the activity of a single concentration of HRP dried and stored on paper was monitored for days SU- was found to stabilize HRP up to times more than trehalose in the short-term ... More
Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents. Less
Biological molecules especially the proteins have a special and important function We study their structure to understand their functions and further make application like the medical research The routine method is diffraction but not work for molecules which cannot grow into crystal and molecules which their crystal are too small Cryo-EM technique provides another way to solve their structures through their images it does not need crystals Meanwhile electron diffraction can work small crystals micro- and nano-crystals after the sample preparation was improved Hence we try to build a method that can restore the structure from the crystal s image ... More
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal�s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Less
Structures of enzyme-substrate product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction Recently using in crystallo catalysis combined with X-ray diffraction we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state By controlling metal ions essential for catalysis the in crystallo approach has revealed unprecedented details of reaction intermediates Here we present protocols used for successful studies of Mg -dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ... More
Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg2+-dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions. Less
Bacteria sense and adapt to environmental changes using two-component systems These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator RR which finally modulates the transcription of target genes The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response NtrX is a modular protein with REC AAA and DNA-binding domains an architecture that classifies it among the NtrC subfamily of RRs However it lacks the signature GAFTGA motif that is essential for activating transcription by ... More
Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA +, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs. Less
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles It is also used to combat infection with pathogenic microbes The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg LC proteins coupled to phosphatidylethanolamine PE on autophagosomal membranes To understand how RavZ recognizes and deconjugates LC -PE we prepared semisynthetic LC proteins and elucidated the structures of the RavZ LC interaction Semisynthetic LC proteins allowed the analysis of structure-function relationships RavZ extracts LC -PE from the membrane before deconjugation RavZ initially recognizes the LC molecule on membranes via its ... More
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy. Less
Angiotensin II receptors AT R and AT R serve as key components of the renin-angiotensin-aldosterone system While AT R plays a central role in the regulation of blood pressure the function of AT R is enigmatic with a variety of reported effects To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors we report crystal structures of the human AT R bound to an AT R-selective and an AT R AT R-dual ligand respectively capturing the receptor in an active-like conformation Unexpectedly helix VIII was found in a non-canonical position stabilizing the active-like state but at ... More
Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands. Less
DNA has been proposed as a highly desirable medium for storage of digital information The barrier to such use of DNA is the low efficiency and speed as well as the high cost of current synthesis methods In the current state of the art DNA is synthesized using phosphoramidite precursors in organic solvents These chemical synthesis methods result in errors of approximately and take approximately minutes per addition step Furthermore the reagents that are used in this synthesis process are expensive Some of these same reagents also damage DNA a problem that precludes the possibility of synthesizing DNA strands that ... More
DNA has been proposed as a highly desirable medium for storage of digital information. The barrier to such use of DNA is the low efficiency and speed as well as the high cost of current synthesis methods. In the current state of the art, DNA is synthesized using phosphoramidite precursors in organic solvents. These chemical synthesis methods result in errors of approximately 1% and take approximately 10 minutes per addition step. Furthermore, the reagents that are used in this synthesis process are expensive. Some of these same reagents also damage DNA, a problem that precludes the possibility of synthesizing DNA strands that are longer than ˜200 bases, further hampering the efficiency of this chemical process. Despite multiple efforts, a feasible method for synthesis of custom nucleic acid sequences using terminal deoxynucleotidyl transferase (TdT) has not been described before. TdT is currently used in batch reactions for the addition of variable lengths of singular nucleotides or uncontrolled sequence of nucleotide mixtures to the 3′ end of a nucleic acid sequence. A method to control the number and nature of nucleotides that TdT incorporates to generate user-defined nucleic acid sequences is a significant challenge which has not been addressed. There thus remains a need for the development of faster and cheaper enzymatic oligonucleotide synthesis methods than the existing chemical oligonucleotide synthesis methods. Less
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids Given the insolubility of the natural C C fatty acyl substrate of the PpsC dehydratase DH domain we investigated its structure function relationships in the presence of shorter surrogate substrates Since most enzymes belonging to the R -specific enoyl hydratase hydroxyacyl dehydratase family conduct the reverse hydration reaction ... More
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28?C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure?function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase?ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme. Less
Insulin-regulated aminopeptidase IRAP is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at resolution In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed forming a hollow structure that excludes external solvent access to the catalytic center A loop adjacent to the enzyme s GAMEN motif undergoes structural reconfiguration allowing the ... More
Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 � resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme�s GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP�s catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness. Less
This multidisciplinary project begins with one overarching aim to elucidate the role of the rigidity of the lipid tail on the phase transitions of lipidic mesophases Previous studies have demonstrated that the position and the number of cis double bonds in monoacylglycerols determine the chain splay of the molecule establishing how this parameter was essential in influencing the phase behavior Following on from this novel lipids which are inspired by naturally occuring cyclopropanated lipids have been synthesized and their phase behavior elucidated The chain rigidity has been systematically varied by locking the cis configuration of the double bond on the ... More
This multidisciplinary project begins with one overarching aim: to elucidate the role of the
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme
During production purification formulation and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability Such harmful conditions include extreme pH changes high ionic strengths or elevated temperatures The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development In this context PEGylation i e the covalent attachment of polyethylene glycol PEG to proteins represents a valuable strategy to improve the physico-chemical properties of proteins In this work the influence ... More
During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2 kDa, 5 kDa and 10 kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved. Less
MEK is an upstream kinase in MAPK signaling pathways where it phosphorylates p MAPK and JNK in response to mitogenic and cellular stress queues MEK is overexpressed and induces metastasis in advanced prostate cancer lesions However the value of MEK as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK have not been developed Despite a high level of sequence homology in the ATP-binding site most reported MEK inhibitors are selective for MEK and display reduced potency toward other MEKs Here we present the first functional and binding selectivity-profiling platform of the MEK family We ... More
MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms. Less
We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening HTS conditions The read time for an entire -well plate is less than min This instrument is particularly well suited for assays based on fluorescence resonance energy transfer FRET Intramolecular protein biosensors with genetically encoded green fluorescent protein GFP donor and red fluorescent protein RFP acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of ... More
We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery. Less
Influenza A haemagglutinin is a surface glycoprotein of Influenza virus responsible for the initial attachment of the virus to the target cell and at a later stage for viral membrane fusion At the acidic pH of the endosome the HA molecule undergoes an irreversible structural rearrangement In consequence the hydrophobic terminal segments of HA are moved to the same end of the refolded molecule promoting membrane fusion haemagglutinin subtypes H -H identified to date can be divided into two groups based on characteristic structural features The low pH-induced structures of proteolytically prepared and E coli-expressed fragments of influenza A H ... More
Influenza A haemagglutinin is a surface glycoprotein of Influenza virus,
responsible for the initial attachment of the virus to the target cell and, at a later
stage, for viral membrane fusion. At the acidic pH of the endosome, the HA
molecule undergoes an irreversible structural rearrangement. In consequence, the
hydrophobic terminal segments of HA2 are moved to the same end of the refolded
molecule, promoting membrane fusion.
16 haemagglutinin subtypes (H1-H16) identified to date can be divided into two
groups based on characteristic structural features. The low pH-induced structures
of proteolytically prepared and E.coli-expressed fragments of influenza A H3 HA2
(group 2 HA) were previously determined by X-ray crystallography.
This study presents structures of proteolytically prepared and recombinantlyexpressed fragments of H1 HA2 in a postfusion conformation. Refolded H1 HA2,
belonging to group 1 HA, adopts a hairpin-like conformation, similar to that of a
rearranged H3 HA2. Structures were compared to the known structures of low pHactivated HA2, to gain a better understanding of the structural differences between
the two groups of HA.
The data show the structures of the refolded HA2 to be conserved between the HA
groups with minor differences.
These structural data are supplemented with functional studies involving the
cross-reactive FI6 antibody. FI6 antibody binds near the conserved fusion
subdomain of the HA molecule and thus interferes with the low pH-triggered
conformational change of HA. Additional methods employed in this study, such as
limited proteolysis, electron microscopy, biolayer interferometry and MDCK1 cell
infection, give insight into the mechanism of FI6 antibody-mediated neutralization,
and highlight the differences in infectivity of H1N1 and H3N2 viruses neutralized
by the FI6 antibody. Less
responsible for the initial attachment of the virus to the target cell and, at a later
stage, for viral membrane fusion. At the acidic pH of the endosome, the HA
molecule undergoes an irreversible structural rearrangement. In consequence, the
hydrophobic terminal segments of HA2 are moved to the same end of the refolded
molecule, promoting membrane fusion.
16 haemagglutinin subtypes (H1-H16) identified to date can be divided into two
groups based on characteristic structural features. The low pH-induced structures
of proteolytically prepared and E.coli-expressed fragments of influenza A H3 HA2
(group 2 HA) were previously determined by X-ray crystallography.
This study presents structures of proteolytically prepared and recombinantlyexpressed fragments of H1 HA2 in a postfusion conformation. Refolded H1 HA2,
belonging to group 1 HA, adopts a hairpin-like conformation, similar to that of a
rearranged H3 HA2. Structures were compared to the known structures of low pHactivated HA2, to gain a better understanding of the structural differences between
the two groups of HA.
The data show the structures of the refolded HA2 to be conserved between the HA
groups with minor differences.
These structural data are supplemented with functional studies involving the
cross-reactive FI6 antibody. FI6 antibody binds near the conserved fusion
subdomain of the HA molecule and thus interferes with the low pH-triggered
conformational change of HA. Additional methods employed in this study, such as
limited proteolysis, electron microscopy, biolayer interferometry and MDCK1 cell
infection, give insight into the mechanism of FI6 antibody-mediated neutralization,
and highlight the differences in infectivity of H1N1 and H3N2 viruses neutralized
by the FI6 antibody. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules This activity is required in many cellular membrane trafficking pathways In this work we have determined a high-resolution structure of an EHD machine in the active state The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane By comparing this active state with a previously determined autoinhibited conformation we can deduce the mechanistic details how recruitment of EHDs ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules. This activity is required in many cellular membrane trafficking pathways. In this work, we have determined a high-resolution structure of an EHD machine in the active state. The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane. By comparing this active state with a previously determined autoinhibited conformation, we can deduce the mechanistic details how recruitment of EHDs to membranes is regulated. A comparison with other membrane-associated molecular machines reveals commonalities and differences in the activation mechanism. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking EHD proteins consist of a dynamin-related GTPase domain a helical domain and a C-terminal Eps -homology EH domain Previous studies have revealed the structure of the EHD dimer Furthermore the N terminal region of EHD was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome suggesting an autoinhibitory role However the molecular mechanisms of membrane binding oligomerization and nucleotide hydrolysis have remained obscure ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. EHD proteins consist of a dynamin-related GTPase domain, a helical domain and a C-terminal Eps15-homology (EH) domain,. Previous studies have revealed the structure of the EHD2 dimer. Furthermore, the N terminal region of EHD2 was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome, suggesting an autoinhibitory role However, the molecular mechanisms of membrane binding, oligomerization and nucleotide hydrolysis have remained obscure. To understand the mechanism of membrane recruitment, the crystal structure of an aminoterminally truncated EHD4 dimer in complex with ATPγS and ADP were determined in this thesis. Compared with the EHD2 structure, the helical domains assume an open conformation featuring a 50° rotation relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), it was shown that the opening aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop region in the GTPase domain undergoes a large rearrangement and creates a new interface that allows oligomerization on membranes. These results suggest that the EHD4 structures represent the active EHD conformation, whereas the EHD2 structure is autoinhibited. A model for the activation and oligomerization of EHD proteins was proposed in which a series of domain rearrangements control membrane recruitment and remodeling in the EHD family. A comparison with other peripheral membrane proteins elucidated common and specific features of this activation mechanism. Less
Type phosphatidic acid phosphatases PAP s can be either soluble or integral membrane enzymes In bacteria integral membrane PAP s play major roles in the metabolisms of glycerophospholipids undecaprenyl-phosphate C -P lipid carrier and lipopolysaccharides By in vivo functional experiments and biochemical characterization we show that the membrane PAP coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate PGP phosphatase of B subtilis We also confirm that this enzyme renamed bsPgpB has a weaker activity on C -PP Moreover we solved the crystal structure of bsPgpB at resolution with tungstate a phosphate analog in the active site ... More
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s. Less
The Haemophilus surface fibril Hsf is an unusually large trimeric autotransporter adhesin TAA expressed by the most virulent strains of H influenzae Hsf is known to mediate adhesion between pathogen and host allowing the establishment of potentially deadly diseases such as epiglottitis meningitis and pneumonia While recent research has suggested that this TAA might adopt a novel hairpin-like architecture the characterization of Hsf has been limited to in silico modelling and electron micrographs with no high-resolution structural data available Here the crystal structure of Hsf putative domain PD is reported at resolution The structure corrects the previous domain annotation by ... More
The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel ‘hairpin-like’ architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the ‘hairpin-like’ hypothesis. Less
In this chapter we describe Long Fragment Read LFR technology a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing WGS The addition of LFR prior to WGS on any high-throughput DNA sequencer e g Complete Genomics Revolocity BGISEQ- Illumina HiSeq etc enables the assignment of single-nucleotide polymorphisms SNPs and other genomic variants onto contigs representing contiguous DNA from a single parent haplotypes with N lengths of up to Mb Importantly this is achieved independent of any parental sequencing data or knowledge of parental haplotypes Further the nature of this method allows for the correction of most amplification sequencing ... More
In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10−9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process. Less
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen For development of vaccines with an increased stability at ambient temperatures manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood This work presents a strategy to examine both the colloidal stability and the remaining biological activity of H N influenza viruses under various conditions after an incubation of days H N phase diagrams were generated for several pH values and different initial H N and NaCl concentrations It was shown ... More
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen. For development of vaccines with an increased stability at ambient temperatures, manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood. This work presents a strategy to examine both, the colloidal stability and the remaining biological activity of H1N1 influenza viruses under various conditions after an incubation of 40 days. H1N1 phase diagrams were generated for several pH values and different initial H1N1 and NaCl concentrations. It was shown that the highest H1N1 recoveries were obtained for pH 6 and that moderate amounts of NaCl are favorable for increased recoveries. In contrast to colloidal stability, the highest remaining HA activity was observed at pH 9. The electrostatic and hydrophobic surface properties of H1N1 were investigated to reveal the mechanisms accounting for the decrease in stability. Secondly, the capability of virus precipitation by polyethylene glycol in combination with determination of surface hydrophobicity was proven to be useful as a predictive tool to rank stability under different conditions. This methodology enables the rapid assessment of aggregation propensity of H1N1 formulations and the influence on the activity of the virus particles and might become a standard tool during the development of vaccine formulations. Less
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals Interestingly phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages Here we demonstrate that peroxidase A from D discoideum DdPoxA is a stable monomeric glycosylated and secreted heme peroxidase with homology to mammalian peroxidases The first crystal structure resolution of a mycetozoan peroxidase of this superfamily shows ... More
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m−1 s−1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense. Less
Membrane-like nanodiscs ND have become an important tool for the cell-free expression solubilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs embedded in NDs would be of high importance for structural biology However despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography In the present work we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso Bacteriorhodopsin BR reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The resulting crystals ... More
Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 Å. The structure of BR was solved at 1.9 Å and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs. Less
Riboswitches are structural RNA elements that are generally located in the ' untranslated region of messenger RNA During regulation of gene expression ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time Here we use femtosecond X-ray free electron laser XFEL pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction We demonstrate this approach by determining four structures of the ... More
Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1�3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of �mix-and-inject� time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes. Less
By interacting with hundreds of protein partners - - proteins coordinate vital cellular processes Phosphorylation of the small heat shock protein HSPB within its intrinsically disordered N-terminal domain activates its interaction with - - ultimately triggering smooth muscle relaxation After analyzing the binding of an HSPB -derived phosphopeptide to - - using isothermal calorimetry and X-ray crystallography we have determined the crystal structure of the complete assembly consisting of the - - dimer and full-length HSPB dimer and further characterized this complex in solution using fluorescence spectroscopy small-angle X-ray scattering and limited proteolysis We show that selected intrinsically disordered regions ... More
By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. Less