1052 Citations
Whirlin is a protein essential to sensory neurons Its defects are responsible for nonsyndromic deafness or for the Usher syndrome a condition associating congenital deafness and progressive blindness This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells The HHD domain of whirlin is the only domain shared by all isoforms but its function remains unknown In this article we report its crystal structure in two distinct conformations a monomeric five-helix bundle similar to the known structure of other HHD ... More
Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five-helix bundle, similar to the known structure of other HHD domains, and a three-helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five-helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain. Less
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers FELs for serial femtosecond crystallography measurements The high X-ray intensity in the focus initiates an explosion of the microjet and sample With the advent of X-ray FELs with megahertz rates the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using nm radiation The operation of gas-dynamic nozzles that ... More
Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments. Less
Automation provides a number of benefits to clinical industrial and research laboratories including elimination of human errors improvement in consistency minimization of contamination increased throughput decreased costs and reduced hands-on time At the same time multiplex assays offer the benefits of more data per sample faster reaction times reduced sample consumption enhanced scalability and a number of additional benefits due to elimination of repetitive steps Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes especially when a large number of samples are being analyzed or when a highly multiplexed assay is being developed Automation can be partially ... More
Automation provides a number of benefits to clinical, industrial, and research laboratories, including elimination of human errors, improvement in consistency, minimization of contamination, increased throughput, decreased costs, and reduced hands-on time. At the same time, multiplex assays offer the benefits of more data per sample, faster reaction times, reduced sample consumption, enhanced scalability, and a number of additional benefits due to elimination of repetitive steps. Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes, especially when a large number of samples are being analyzed, or when a highly multiplexed assay is being developed. Automation can be partially or fully implemented. Less
Rotaviruses RVs cause life-threatening diarrhea in infants and children worldwide Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens HBGA as both cell attachment and susceptibility factors for the globally dominant P P and P genotypes of human RVs How these genotypes interact with HBGA is not known Here our crystal structures of P and a neonate-specific P VP s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs consistent with their prevalence Remarkably the VP ... More
Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut. Less
Angiotensin II AngII plays a central role in regulating human blood pressure which is mainly mediated by interactions between AngII and the G-protein-coupled receptors GPCRs AngII type receptor AT R and AngII type receptor AT R We have solved the crystal structure of human AT R binding the peptide ligand Sar Ile AngII and its specific antibody at - resolution Sar Ile AngII interacts with both the core binding domain where the small-molecule ligands of AT R and AT R bind and the extended binding domain which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor We ... More
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs. Less
The chemokine receptor CCR is an important anti-HIV human immunodeficiency virus drug target owning to its pivotal role in HIV- viral entry as a co-receptor Here we present a resolution crystal structure of CCR bound to PF- a second-generation oral CCR antagonist currently in phase II clinical trials PF- and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino N - and carboxyl C - substituents Comparison of the CCR PF- structure with the previously determined structure of CCR in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes ... More
The chemokine receptor CCR5 is an important anti-HIV (human immunodeficiency virus) drug target owning to its pivotal role in HIV-1 viral entry as a co-receptor. Here, we present a 2.9 Å resolution crystal structure of CCR5 bound to PF-232798, a second-generation oral CCR5 antagonist currently in phase II clinical trials. PF-232798 and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino (N)- and carboxyl (C)- substituents. Comparison of the CCR5–PF-232798 structure with the previously determined structure of CCR5 in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes of the receptor. Our results not only offer insights into the phenomenon that PF-232798 has higher affinity and alternative resistance profile to maraviroc, but also will facilitate the design of new anti-HIV drugs. Less
CCR is the primary chemokine receptor utilized by HIV to infect leukocytes whereas CCR ligands inhibit infection by blocking CCR engagement with HIV gp To guide the design of improved therapeutics we solved the structure of CCR in complex with chemokine antagonist P CCL Several structural features appeared to contribute to the anti-HIV potency of P CCL including the distinct chemokine orientation relative to the receptor the near-complete occupancy of the receptor binding pocket the dense network of intermolecular hydrogen bonds and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc Molecular modeling indicated that HIV gp mimicked ... More
CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases. Less
The Machine Recognition of Crystallization Outcomes MARCO initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups Here state-of-the-art machine learning algorithms are trained and tested on different parts of this data set We find that more than of the test images can be correctly labeled irrespective of their experimental origin Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments this approach opens the door to both industrial and fundamental research applications
The bacterium Streptococcus pneumoniae the pneumococcus is a major human pathogen that requires Zn for its survival and virulence in the host environment Polyhistidine triad protein D PhtD has a known role in pneumococcal Zn homeostasis However the mechanistic basis of PhtD function remains unclear partly due to a lack of structural information Here we determined the crystal structure of the fragment PhtD - containing the third Zn -binding histidine triad HT motif of the protein Analysis of the structure suggests that Zn binding occurs at the surface of the protein and that all five HT motifs in the protein ... More
The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition. Less
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality However we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity which is critically required to enable rational design of degraders Here we utilize comprehensive characterization of the ligand dependent CRBN BRD interaction to demonstrate that binding between proteins that have not evolved to interact is plastic Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations which are selectively bound by ligands We demonstrate that computational protein-protein docking can ... More
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands. Less
Type-A -aminobutyric acid GABAA receptors are pentameric ligand-gated ion channels pLGICs typically consisting of subunit combinations They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs such as benzodiazepines BZDs used to treat epilepsy insomnia anxiety panic disorder and muscle spasm However the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown Here we report the cryo-EM structure of a human GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator PAM The receptor subunits assume a unified quaternary activated conformation ... More
Type-A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of α/β/γ subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human α1β3γ2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered α5 and α5γ2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation. Less
-Galactosidases EC are retaining glycosidases that cleave terminal -linked galactose residues from glycoconjugate substrates -Galactosidases take part in the turnover of cell wall associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals Deficiency of human -galactosidase A -Gal A causes Fabry disease FD a heritable X-linked lysosomal storage disorder characterized by accumulation of globotriaosylceramide Gb and globotriaosylsphingosine lyso-Gb Current management of FD involves enzyme-replacement therapy ERT An activity-based probe ABP covalently labeling the catalytic nucleophile of -Gal A has been previously designed to study -galactosidases for use in FD therapy Here we report that this ABP ... More
α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Less
The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds or thousands of individual crystallization trials each of which must be repeatedly examined for crystals or hints of crystallinity Currently the only real way to address this bottleneck is to use an automated imager to capture images of the trials However the images still need to be assessed for crystals or other outcomes Ideally there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result However as yet no such tool exists in wide usage despite ... More
The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds (or thousands) of individual crystallization trials, each of which must be repeatedly examined for crystals or hints of crystallinity. Currently, the only real way to address this bottleneck is to use an automated imager to capture images of the trials. However, the images still need to be assessed for crystals or other outcomes. Ideally, there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result. However, as yet no such tool exists in wide usage, despite this being a well recognized problem. One of the issues in creating robust automatic image-analysis software is the lack of reliable data for training machine-learning algorithms. Here, a mobile application, Cinder, has been developed which allows crystallization images to be scored quickly on a smartphone or tablet. The Cinder scores are inserted into the appropriate table in a crystallization database and are immediately available to the user through a more sophisticated web interface, allowing more detailed analyses. A sharp increase in the number of scored images was observed after Cinder was released, which in turn provides more data for training machine-learning tools. Less
Platelet-activating-factor receptor PAFR responds to platelet-activating factor PAF a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects PAFR is considered an important drug target for treating asthma inflammation and cardiovascular diseases Here we report crystal structures of human PAFR in complex with the antagonist SR and the inverse agonist ABT- at - and - resolution respectively The structures supported by molecular docking of PAF provide insights into the signal-recognition mechanisms of PAFR The PAFR SR structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by and respectively and helix VIII ... More
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR–SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors. Less
The HECT E ligases ubiquitinate numerous transcription factors and signaling molecules and their activity must be tightly controlled to prevent cancer immune disorders and other diseases In this study we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities Biochemical structural and cellular analysis has revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications but complete removal of the brake can ... More
The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analysis has revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self-destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior. Less
MicroED structure of the NaK ion channel reveals a Na+ partition process into the selectivity filter
Sodium Na is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation signaling and facilitation of various transporters The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels yet they are rarely captured by conventional structural biology means Here we use the emerging cryo-electron microscopy cryoEM method micro-electron diffraction MicroED to study the structure of a prototypical tetrameric Na -conducting channel NaK to resolution from nano-crystals Two new conformations at the external site of NaK are identified allowing us to visualize a partially ... More
Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics. Less
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates Solution conditions affect morphologic features such as crystal size as well as kinetic features such as crystal growth time Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols These techniques have limitations in terms of handling large data sets comprehensiveness or completeness To eliminate these limitations morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have ... More
Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphologic features, such as crystal size, as well as kinetic features, such as crystal growth time. Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols. These techniques have limitations in terms of handling large data sets, comprehensiveness or completeness. To eliminate these limitations, morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram method. Morphologic features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength, and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the empirical phase diagram method can support high-throughput crystallization experiments in its data amount as well as its data complexity. Less
Structure-based drug design SBDD heavily relies on the production of high-resolution three-dimensional D structures of the drug target in the presence or absence of the drug candidate X-ray crystallography is the predominant technique accounting for higher than of the structures in Protein Data Bank PDB considering the complexes involving protein and inhibitors Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process It is the main technique used to obtain D information for binary complexes involving ... More
Structure-based drug design (SBDD) heavily relies on the production of high-resolution (<2Å) three-dimensional (3D) structures of the drug target in the presence or absence of the drug candidate [1]. X-ray crystallography is the predominant technique accounting for higher than 94% of the structures in Protein Data Bank (PDB), considering the complexes involving protein and inhibitors [2–4]. Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process [5]. It is the main technique used to obtain 3D information for binary complexes involving protein and drugs [6]. One of the major ‘bottlenecks’ in X-ray crystallography is the lack of generalized methods for high quality crystal production. Since protein crystallization mechanism details remain unknown, protein crystallization is a complicated and time-consuming process and requires performing a significant number of trial-and-error experiments involving systematic testing of variable chemical and physical parameters [7]. High-throughput (HT) structural biology coincided with the dawn of the genomic era in biology requires the automation, miniaturization, and parallelization of protein crystallization in order to reach the capacity necessary for large-scale structure determination efforts [8]. HT protein crystallization screening (HTPCS) technologies appeared on the protein crystallization scene more than three decades ago and have since allowed accessing hundreds to thousands of protein crystallization conditions, thereby greatly impacting HT structural biology [9]. HTPCS has helped to identify critical components required for HT crystallization efforts [7]. In recent years, the rapid developments of manipulation techniques and devices provide effective and reliable solutions for protein crystallization screening with HT and low consumption. In spite of their advances, HTPCS has suffered from two main handicaps, namely, poor hit rate in protein crystallization screening and lack of predictive power of the scoring functions. To overcome these handicaps, several projects have recently been initiated to construct ‘smart systems’ that are not only capable of rapidly performing a large number of crystallization trials, but also scripting and triggering certain events based on the collected data used in predict the outcome of a protein x-ray crystallization trial [10]. In this contest, we will outline recent efforts in HTPCS that could improve the success rate of the structural pipeline. We will discuss the challenge and some of the possible avenues in that direction. Less
Neuropeptide Y NPY receptors belong to the G protein-coupled receptor GPCR superfamily and play important roles in food intake anxiety and cancer regulation The NPY Y receptor system has emerged as one of the most complex networks with three peptide ligands NPY peptide YY and pancreatic polypeptide binding to four receptors in mammals namely Y Y Y and Y receptors with different affinity and selectivity NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y receptor Y R A number of peptides and small-molecule compounds have been characterized as Y R antagonists and ... More
Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors. Less
Epstein-Barr virus EBV is a causative agent of infectious mononucleosis and is associated with new cases of cancer and deaths annually Subunit vaccines against this pathogen have focused on the gp glycoprotein and remain unsuccessful We isolated human antibodies recognizing the EBV fusion machinery gH gL and gB from rare memory B cells One anti-gH gL antibody AMMO potently neutralized infection of B cells and epithelial cells the two major cell types targeted by EBV We determined a cryo-electron microscopy reconstruction of the gH gL-gp -AMMO complex and demonstrated that AMMO bound to a discontinuous epitope formed by both gH ... More
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells; the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden. Less
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein related lipid transfer StART domains that are implicated in intracellular non-vesicular lipid transport A new family of membrane-anchored StARkins was recently identified including six members Lam Lam in the yeast Saccharomyces cerevisiae Lam Lam are anchored to the endoplasmic reticulum ER membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast To better understand the biological roles of these proteins we carried out a structure-function analysis of the second StARkin domain of Lam here termed Lam S NMR ... More
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein–related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1–Lam6, in the yeast Saccharomyces cerevisiae. Lam1–Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling “head-first” entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress. Less
Mitochondrial Carriers MCs are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism The ADP ATP Carriers AACs are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP In human four different AACs isoforms are described which are expressed in tissue-specific manner They are involved in different genetic diseases and play a role in cancerogenesis Up to now only the structures of the bovine isoform and yeast isoforms and AAC have been determined in one particular conformation obtained in complex ... More
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded. Less
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum Pf circumsporozoite protein PfCSP from several subjects immunized with an attenuated Pf whole-sporozoite SPZ vaccine Sanaria PfSPZ Vaccine Passive transfer of one of these antibodies monoclonal antibody CIS conferred high-level sterile protection in two different mouse models of malaria infection The affinity and stoichiometry of CIS binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain The first ... More
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design. Less
As a protective envelope surrounding the bacterial cell the peptidoglycan sacculus is a site of vulnerability and an antibiotic target Peptidoglycan components assembled in the cytoplasm are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate A product of peptidoglycan synthesis undecaprenyl-pyrophosphate is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase BacA To understand how BacA functions we determine its crystal structure at resolution The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane Conserved residues map to the pocket where pyrophosphorolysis occurs BacA ... More
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase. Less
Investigation of the biochemical basis of life is a long standing challenge for scientists The field of molecular biology was established based on the pioneering studies of macromolecular structures including the structural characterization of the DNA double helix and the description of the first protein structures These early achievements along with the technical development e g automatic data collection and computer-assisted data analysis enables extremely efficient and fast determination of structure and dynamic properties of proteins
High-throughput single-cell RNA-seq methods assign limited unique molecular identifier UMI counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts We thus developed a high-throughput single-cell RNA-seq method Quartz-Seq to overcome these issues Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts at a rate of and detect more genes To demonstrate the power of Quartz-Seq we analyzed approximately transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads
The low limits of detection afforded by second harmonic generation SHG microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients APIs within amorphous solid dispersions ASDs ASDs in which an API is maintained in an amorphous state within a polymer matrix are finding increasing use to address solubility limitations of small-molecule APIs Extensive stability testing is typically performed for ASD characterization the time frame for which is often dictated by the earliest detectable onset of crystal formation Here a study of accelerated stability testing on ritonavir a human immunodeficiency virus HIV protease ... More
The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 �C/75%RH and 40 �C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined. Less
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the -oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA RNA production This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring To provide validation for this approach as a drug target we have RNAi silenced the three -oxopurine phosphoribosyltransferase PRTase isoforms in the infectious stage of Trypanosoma brucei demonstrating that ... More
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery. Less
Plastidial thioredoxin TRX -like proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs we solved the crystal structures of poplar TRX-like in oxidized and reduced states and of mutated variants These structures share similar folding with TRXs exhibiting the canonical WCGPC signature Moreover the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C S C S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins Modeling of the ... More
Plastidial thioredoxin (TRX)-like2.1 proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling. To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs, we solved the crystal structures of poplar TRX-like2.1 in oxidized and reduced states and of mutated variants. These structures share similar folding with TRXs exhibiting the canonical WCGPC signature. Moreover, the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C45S/C67S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins. Modeling of the interaction of TRX-like2.1 with both NADPH- and ferredoxin-thioredoxin reductases (FTR) indicates that the presence of Arg43 and Lys44 residues likely precludes reduction by the plastidial FTR. Less
Methods are provided for analyzing one or more genetic samples comprising procuring one or more genetic samples comprising genetic material from one or more individuals and sequencing the genetic material using non-targeted ultra-low coverage sequencing to obtain genetic information for individual associated with the one or more genetic samples Personal and genetic information associated with the individuals is stored in a database for retrieval and manipulation
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains ECDs comparable to the water channels nm of the mesophase Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid Ia d double-diamond Pn m and double-primitive Im m space groups with water channels five times larger than traditional lipidic mesophases and showing re-entrant behavior upon increasing hydration of sequences Ia d ... More
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3�5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d?Pn3m?Ia3d and Pn3m?Im3m?Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology. Less
Cells require some metals such as zinc and manganese but excess levels of these metals can be toxic As a result cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication Here we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation Altogether we identified genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment and putative zinc-responsive genes Analysis of the omics data points to ... More
Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the ‘omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the ‘omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes. Less
Drugs frequently require interactions with multiple targets via a process known as polypharmacology to achieve their therapeutic actions Currently drugs targeting several serotonin receptors including the -HT C receptor are useful for treating obesity drug abuse and schizophrenia The competing challenges of developing selective -HT C receptor ligands or creating drugs with a defined polypharmacological profile especially aimed at G protein-coupled receptors GPCRs remain extremely difficult Here we solved two structures of the -HT C receptor in complex with the highly promiscuous agonist ergotamine and the -HT A-C receptor-selective inverse agonist ritanserin at resolutions of and respectively We analyzed their ... More
Drugs frequently require interactions with multiple targets — via a process known as polypharmacology — to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin, at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs. Less
Canonical FGFs activate FGF receptors FGFR via paracrine or autocrine mechanisms in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation By contrast endocrine FGFs FGF and are circulating hormones that regulate critical metabolic processes in a variety of tissues FGF regulates bile acid synthesis and lipogenesis while FGF stimulate insulin sensitivity energy expenditure and weight loss Endocrine FGFs signal through FGFRs in a manner that requires Klothos which are cell surface proteins with tandem glycosidase domains Here we describe the crystal structures of free and ligand-bound -Klotho extracellular regions revealing the molecular mechanism ... More
Canonical FGFs activate FGF receptors (FGFR) via paracrine or autocrine mechanisms, in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation1,2. By contrast, endocrine FGFs (FGF 19, 21, and 23) are circulating hormones that regulate critical metabolic processes in a variety of tissues3,4. FGF19 regulates bile acid synthesis and lipogenesis, while FGF21 stimulate insulin sensitivity, energy expenditure and weight loss5. Endocrine FGFs signal through FGFRs in a manner that requires Klothos, which are cell surface proteins with tandem glycosidase domains3,4. Here we describe the crystal structures of free and ligand-bound β-Klotho extracellular regions, revealing the molecular mechanism underlying the specificity of FGF21 towards β-Klotho and demonstrating how FGFR is activated in a Klotho-dependent manner. β-Klotho serves as a primary “Zip code”-like receptor for FGF21 with an FGFR functioning as a catalytic subunit that mediates intracellular signaling. Our structures also show how a sugar cutting enzyme (glycosidase) has evolved to become a specific receptor for hormones that regulate metabolic processes including lowering of blood sugar. Finally, we describe a superior agonistic variant of FGF21 and present structural insights offering development of novel therapeutics for diseases linked to endocrine FGFs. Less
The ageing suppressor -klotho binds to the fibroblast growth factor receptor FGFR This commits FGFR to respond to FGF a key hormone in the regulation of mineral ion and vitamin D homeostasis The role and mechanism of this co-receptor are unknown Here we present the atomic structure of a ternary complex that consists of the shed extracellular domain of -klotho the FGFR c ligand-binding domain and FGF In this complex -klotho simultaneously tethers FGFR c by its D domain and FGF by its C-terminal tail thus implementing FGF FGFR c proximity and conferring stability Dimerization of the stabilized ternary complexes ... More
The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23–FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling. Less
The maturation of Ras GTPases and other cellular CaaX proteins involves three enzymatic steps addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine C in the C-terminal CaaX motif proteolytic cleavage of the aaX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase ICMT a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum ER ICMT is the only cellular enzyme known to methylate prenylcysteine substrates methylation is important for their biological functions including the membrane localisations and subsequent activities of Ras prelamin A and Rab ICMT ... More
The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes. Less
Class B G-protein-coupled receptors GPCRs which consist of an extracellular domain ECD and a transmembrane domain TMD respond to secretin peptides to play a key part in hormonal homeostasis and are important therapeutic targets for a variety of diseases Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket Recently three structures of class B GPCRs in complex with peptide ligands have been solved These structures provide essential ... More
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases1,2,3,4,5,6,7,8. Previous work9,10,11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved12,13,14. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs. Less
In recent years highly detailed characterization of adult bone marrow BM myeloid progenitors has been achieved and as a result the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined Fetal liver FL hematopoietic progenitor cells HPCs are poorly characterized in comparison potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis Numerous disorders for example infant acute leukemias have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets We previously demonstrated that a Runx distal promoter P -GFP proximal promoter P -hCD ... More
In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible. Less
Protein crystallography has significantly advanced in recent years with in situ data collection in which crystals are placed in the X-ray beam within their growth medium being a major point of focus In situ methods eliminate the need to harvest crystals a previously unavoidable drawback particularly for often small membrane-protein crystals Here we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature C and under cryogenic conditions The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive easy to make and compatible with automated ... More
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min. Less
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle and are thus drug targets for the treatment of insomnia Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source we determined structures of human orexin receptor in complex with the subtype-selective antagonist EMPA N-ethyl- - -methoxy-pyridin- -yl - toluene- -sulfonyl -amino -N-pyridin- -ylmethyl-acetamide at - and - resolution In comparison with the non-subtype-selective antagonist suvorexant EMPA contacted fewer residues through hydrogen bonds at the orthosteric site explaining the faster dissociation rate Comparisons among these OX R structures in complex with selective antagonists and ... More
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Less
A licensed vaccine for respiratory syncytial virus RSV is unavailable and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants Recently isolated antibodies C and D are substantially more potent than palivizumab and a derivative of D is in clinical trials Here we show that unlike D C preferentially neutralizes subtype A viruses The crystal structure of C bound to the RSV fusion F protein reveals that the overall binding mode of C is similar to that of D but their angles of approach are substantially different Mutagenesis and virological studies demonstrate that RSV F residue is largely ... More
A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines. Less
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task However by a systematic screening approach the time to obtain suitable ... More
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties. Less
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism signaling and ion homeostasis About half of all drug targets are membrane proteins Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics However the pace of IMP crystallography has been relatively slow due to great challenges in crystallization Lipid cubic phase LCP has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment ... More
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism, signaling, and ion homeostasis. About half of all drug targets are membrane proteins. Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics. However, the pace of IMP crystallography has been relatively slow due to great challenges in crystallization. Lipid cubic phase (LCP) has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment. Its bilayer is composed of neutral lipids, such as monoacylglycerols, and can accommodate a substantial amount of native lipids such as phospholipids and cholesterol. Thus, the structure and composition of LCP mimic biomembranes and therefore offer a native-like environment for membrane proteins, which is favorable for functionality and crystallization. Here, the principles for LCP formation, membrane protein reconstitution, and crystallization process are described. The successful application of LCP crystallization for a wide range of membrane proteins including receptors, complexes, transporters, channels, enzymes, membrane protein insertion chaperons, and outer membrane �-barrels is summarized. General methods and protocols for this method are also described. Less
Ferredoxin-dependent bilin reductases FDBRs are a class of enzymes reducing the heme metabolite biliverdin IX BV to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms Thus far seven FDBR families have been identified each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate The newest addition to the family is PcyX originally identified from metagenomics data derived from phage Phylogenetically PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin PcyX however converts the same substrate to phycoerythrobilin resembling the reaction catalysed by cyanophage PebS Within this study ... More
Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. Less
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders NSDs Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms However traditional assays that track complex neuronal phenotypes such as neuronal connectivity exhibit poor scalability and are not compatible with high-throughput screening HTS procedures Therefore we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes First using inexpensive laboratory-level automation we industrialized primary neuronal culture production which enabled the creation ... More
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders. Less
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection Peroxide stress regulators PerRs are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites PerR binding to gene promoters is controlled by the presence of iron in the regulatory site and iron-catalyzed oxidation of PerR by H O leads to the dissociation of PerR from DNA In addition to a regulatory metal most PerRs require a structural metal for proper dimeric assembly We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans a rare example ... More
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA–binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress. Less
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens often through irreversible damage of their cellular membranes To explore the mechanism by which the important cathelicidin peptide LL- of the human innate immune system interacts with membranes we performed biochemical biophysical and structural studies The crystal structure of LL- displays dimers of anti-parallel helices and the formation of amphipathic surfaces Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface Furthermore hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents Both scaffolds underline ... More
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics. Less
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins Recombinant proteins are biological macromolecules which are produced using genetically modi ed organisms GMOs In addition to the food industry and for the synthesis of organic compounds industrially produced proteins are mainly used in the eld of medicine There they make a decisive contribution to the diagnosis prevention and treatment of various human diseases The production of protein-based drugs biopharmaceuticals and their provision in a stable and bioavailable dosage form formulation are a challenge due to the size and complexity of these ... More
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins. Recombinant proteins are biological macromolecules, which are produced using genetically modi ed organisms (GMOs). In addition to the food industry and for the synthesis of organic compounds, industrially produced proteins are mainly used in the eld of medicine. There, they make a decisive contribution to the diagnosis, prevention and treatment of various human diseases. The production of protein-based drugs (biopharmaceuticals) and their provision in a stable and bioavailable dosage form (formulation) are a challenge due to the size and complexity of these molecules. On the way to the nal drug product, the target protein undergoes a long and complex processing chain consisting of GMO cultivation (upstream processing), puri - cation operations (downstream processing) and formulation steps. The reaction medium for the entire manufacturing process is mainly based on aqueous solutions. Some of the process steps used require extreme conditions such as unphysiological salt concentrations (e.g. in chromatographic puri cation) or acidic pH values (e.g. virus inactivation); all of which are potential stress factors for the protein integrity. Resulting irreversible changes in protein structure and physical instabilities, e.g. aggregation can a ect both drug safety and ecacy. In order to ensure the product quality during the entire manufacturing process, the development of strategies for the stabilization of proteins in aqueous solutions is of paramount importance. From an economic perspective, short development times are desirable in order to reduce the `time to market' of a drug product. Standardized procedures are of great interest for shortening the development times of stabilized protein products and are therefore the subject of this dissertation. Less
Deubiquitinating enzymes DUBs have garnered significant attention as drug targets in the last years The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins especially proteins that are challenging to directly target but which are stabilized by DUB family members Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools Most reported DUB inhibitors however are polypharmacological agents possessing weak micromolar potency toward their primary target limiting their utility in target validation and mechanism studies Due to a lack of high-resolution DUB small-molecule ligand complex structures no structure-guided ... More
Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. Less
CLYBL encodes a ubiquitously expressed mitochondrial enzyme conserved across all vertebrates whose cellular activity and pathway assignment are unknown Its homozygous loss is tolerated in seemingly healthy individuals with reduced circulating B levels being the only and consistent phenotype reported to date Here by combining enzymology structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells Cells lacking CLYBL accumulate citramalyl-CoA an intermediate in the C -dicarboxylate metabolic pathway that includes itaconate a recently identified human antimicrobial metabolite and immunomodulator We report that CLYBL loss leads to a cell autonomous defect in the ... More
CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. We report that CLYBL loss leads to a cell autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation. Less