1194 Citations
Studies were undertaken to assess the merits and limitations of second-harmonic generation SHG for the selective detection of protein and polypeptide crystal formation focusing on the potential for false positives from SHG-active salts present in crystallization media The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples Out of salts investigated six produced significant background SHG and of the wells of a sparse-matrix screen produced SHG upon solvent evaporation SHG-active salts include phosphates hydrated sulfates formates and tartrates while chlorides acetates and anhydrous sulfates resulted in no detectable SHG activity The identified ... More
Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants. Less
Bacterial DsbA enzymes catalyze oxidative folding of virulence factors and have been identified as targets for antivirulence drugs However DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA EcDsbA Nonetheless sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA For example Salmonella enterica typhimurium SeDsbA sequence identity to EcDsbA shares almost identical structural surface and redox properties Using comparative sequence and structure analysis ... More
Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class. Less
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained This methodology is time consuming and resource intensive hindering protein structure determination Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I PSI due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements Structure determination techniques tailored for these difficult to crystallize proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics Here we demonstrate a simple and robust ... More
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these �difficult to crystallize� proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. Less
The Toll interleukin- receptor TIR domain is a protein protein interaction domain that is found in both animal and plant immune receptors The N-terminal TIR domain from the nucleotide-binding NB leucine-rich repeat LRR class of plant disease-resistance R proteins has been shown to play an important role in defence signalling Recently the crystal structure of the TIR domain from flax R protein L was determined and this structure combined with functional studies demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L To advance the molecular understanding of the function of TIR domains in R-protein signalling ... More
The Toll/interleukin-1 receptor (TIR) domain is a protein–protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)–leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 Å resolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 Å resolution, had the symmetry of space group P41212 or P43212 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 Å resolution and had the symmetry of space group P41212 or P43212, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity. Less
Current HIV- vaccines elicit strain-specific neutralizing antibodies However cross-reactive neutralizing antibodies arise in of HIV- -infected individuals and details of their generation could provide a roadmap for effective vaccination Here we report the isolation evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection The mature antibody CH neutralized of HIV- isolates and its co-crystal structure with gp revealed a novel loop-based mechanism of CD -binding site recognition Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation Notably the CH -lineage unmutated common ancestor avidly bound the transmitted founder HIV- ... More
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in ~20% of HIV-1-infected individuals, and details of their generation could provide a roadmap for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection. The mature antibody, CH103, neutralized ~55% of HIV-1 isolates, and its co-crystal structure with gp120 revealed a novel loop-based mechanism of CD4-binding site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the CH103-lineage unmutated common ancestor avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data elucidate the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies and provide insights into strategies to elicit similar antibodies via vaccination. Less
Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals thus overcoming the experimental riddle faced in traditional crystallography methods Here we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations Crystals ... More
Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography. Less
Mechanosensitive channels MS are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture Therefore these channels serve as emergency valves when experiencing significant environmental stress The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein So far archeal mechanosensitive channels of small conductance have ... More
Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins. Less
Aims The prototypical protein disulfide bond Dsb formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli However genomic analysis has revealed several distinct Dsb-like systems in bacteria including the pathogen Salmonella enterica serovar Typhimurium This includes the scsABCD locus which encodes a system that has been shown via genetic analysis to confer copper tolerance but whose biochemical properties at the protein level are not defined The aim of this study was to provide functional insights into the soluble ScsC protein through structural biochemical and genetic analyses Results Here we ... More
Aims: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. Results: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of �-sheets and a-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. Innovation and Conclusion: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity. Antioxid. Redox Signal. 19, 1494�1506. Less
The plant hormones cytokinins play a central role in regulating cell division and developmental events Cytokinin oxidase regulates the levels of these plant hormones by catalyzing their irreversible oxidation which contributes to the regulation of various morpho-physiological processes controlled by cytokinins In this study the crystallization and preliminary X-ray diffraction analysis of the flax cytokinin oxidase LuCKX are reported Plate-like crystals of LuCKX were obtained using PEG as a precipitant and diffracted X-rays to resolution The protein crystals have the symmetry of space group C and are most likely to contain two molecules per asymmetric unit
In mammals Toll-like receptors TLRs recognize conserved microbial molecular signatures and induce an early innate immune response in the host TLR signalling is mediated by interactions between the cytosolic TIR Toll interleukin- receptor domains of the receptor and the adaptor proteins Increasingly it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis Studies have shown that TcpB interferes with the TLR and TLR signalling pathways to inhibit TLR-mediated inflammatory responses Such interference may involve TIR TIR-domain interactions between bacterial and mammalian ... More
In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR�TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120�250) are reported. The crystals diffracted to 2.6 � resolution, have the symmetry of the monoclinic space group P21 and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host. Less
The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors The archetypal disulfide oxidase proteins in Escherichia coli Ec are DsbA and DsbB which together form a functional unit DsbA introduces disulfides into folding proteins and DsbB re oxidizes DsbA to maintain it in the active form In Mycobacterium tuberculosis Mtb no DsbB homologue is encoded but a functionally similar but structurally divergent protein MtbVKOR has been identified Here the Mtb protein Rv c is investigated and it is shown that it is the DsbA-like partner ... More
The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB re�oxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity. Less
Nuclear pore complexes NPCs are giant molecular machines embedded in the double membrane that surrounds the eukaryotic nucleus Deciphering the molecular structure of the NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport In less than a decade atomic level structures of many nucleoporins Nups have been solved and the molecular picture of the NPC is becoming increasingly clearer Nup subcomplex is a nucleoporin subcomplex in the NPC s central channel the molecular structure of which is not known so far At a sequence level the N-terminal half of all subunits in the ... More
Nuclear pore complexes (NPCs) are giant molecular machines embedded in the double
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold. Less
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold. Less
With increasingly large immunocompromised populations around the world opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality To combat the paucity of antifungal compounds new drug targets must be investigated Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate Although the enzyme is ubiquitous and well characterized in other kingdoms no crystallographic studies on the fungal protein have been performed Presented here are the expression purification crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase The crystals had the symmetry ... More
With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P212121 and diffracted to 2.2 � resolution. Less
In higher plants the MADS-box genes encode a large family of transcription factors TFs involved in key developmental processes most notably plant reproduction flowering and floral organ development SEPALLATA SEP is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs SEP is divided into four domains the M MADS domain involved in DNA binding and dimerization the I intervening domain a short domain involved in dimerization the K keratin-like domain important for multimeric MADS complex formation and the C C-terminal domain ... More
In higher plants, the MADS-box genes encode a large family of transcription factors (TFs) involved in key developmental processes, most notably plant reproduction, flowering and floral organ development. SEPALLATA 3 (SEP3) is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs. SEP3 is divided into four domains: the M (MADS) domain, involved in DNA binding and dimerization, the I (intervening) domain, a short domain involved in dimerization, the K (keratin-like) domain important for multimeric MADS complex formation and the C (C-terminal) domain, a largely unstructured region putatively important for higher-order complex formation. The entire K domain along with a portion of the I and C domains of SEP3 was crystallized using high-throughput robotic screening followed by optimization. The crystals belonged to space group P21212, with unit-cell parameters a = 123.44, b = 143.07, c = 49.83 �, and a complete data set was collected to 2.53 � resolution. Less
During the past year electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope There have also been key developments in software and in molecular replacement and phase extension methods designed to facilitate the process of structure determination
Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals In this context the present study reports the isolation recombinant expression biochemical and structural characterization of a novel endoxylanase family GH SCXyl identified from sugarcane soil metagenome The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH and C The crystal structure was solved at resolution revealing the classical a -barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp -Arg loop that can adopt distinct conformational states ... More
Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45�C. The crystal structure was solved at 2.75 � resolution, revealing the classical (�/a)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. Less
Nonlinear optical NLO instrumentation has been integrated with synchrotron X-ray diffraction XRD for combined single-platform analysis initially targeting applications for automated crystal centering Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning Two optical designs were constructed and characterized one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer Both instruments enabled protein crystal identification with integration times between and s per pixel representing a -fold reduction in the per-pixel exposure time relative to X-ray raster scanning Quantitative centering and analysis of phenylalanine hydroxylase ... More
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. Less
As part of the mammalian innate immune response Toll-like receptors and can signal via the adaptor protein TRIF TICAM- to elicit the production of type-I interferons and cytokines Recent studies have suggested an auto-inhibitory role for the N-terminal domain NTD of TRIF This domain has no significant sequence similarity to proteins of known structure In this paper the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA M L M are reported Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol as precipitant diffracted X-rays to resolution To facilitate phase determination two additional methionines were incorporated ... More
As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 � resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 � resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P212121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain. Less
Autophagy is a degradation pathway conserved in eukaryotes Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content After fusion of the membrane an autophagosome vesicle is formed which then fuses with the vacuole or lysosome where its content is degraded PROPPINs -propeller proteins that bind polyphosphoinositides play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif The three yeast PROPPINs Atg Atg and Hsv are involved in different subtypes of autophagy In this study I purified different Atg Atg and Hsv PROPPIN homologs and showed that they bind ... More
Autophagy is a degradation pathway conserved in eukaryotes. Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content. After fusion of the membrane, an autophagosome vesicle is formed, which then fuses with the vacuole (or lysosome) where its content is degraded. PROPPINs, �-propeller proteins that bind polyphosphoinositides, play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif. The three yeast PROPPINs Atg18, Atg21 and Hsv2 are involved in different subtypes of autophagy.
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields Less
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields Less
Structural studies of integral membrane proteins IMPs are often hampered by difficulties in producing stable homogenous samples for crystallization To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization For such an approach to be effective an efficient screening strategy is imperative To this end strategies have been developed that involve the use of green fluorescent protein GFP fusion constructs However these approaches suffer from two drawbacks proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require ... More
Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning. Less
Introduction X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution It provides key information for the understanding of protein function opening opportunities for the modulation of enzymatic mechanisms and protein ligand interactions As a consequence macromolecular crystallography plays an essential role in drug design as well as in the a posteriori validation of drug mechanisms Areas covered The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past years As a consequence access to the facilities required for these investigations such as synchrotron beamlines became more difficult and significant efforts ... More
Introduction: X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein�ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms.
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
The smoothened SMO receptor a key signal transducer in the Hedgehog Hh signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis The SMO receptor is classified as a class Frizzled class F G protein-coupled receptor GPCR although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than Here we report the crystal structure at resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY Although the SMO receptor shares the seven transmembrane helical TM fold ... More
The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. Less
The potential of second-harmonic generation SHG microscopy for automated crystal centering to guide synchrotron X- ray diffraction of protein crystals was explored These studies included i comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and ii X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging In studies using adrenergic receptor membrane-protein crystals prepared in lipidic mesophase the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X- ray minibeam ... More
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-�ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using �2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-�ray minibeam. SHG imaging was found to provide about 2 �m spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed. Less
Dynamin -like protein DNM L mediates fission of mitochondria and peroxisomes and dysfunction of DNM L has been implicated in several neurological disorders To study the molecular basis of mitochondrial remodelling we determined the crystal structure of DNM L that is comprised of a G domain a bundle signalling element and a stalk DNM L assembled via a central stalk interface and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM L on membranes and its ... More
Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria. Less
When protein crystals are submicrometre-sized X-ray radiation damage precludes conventional diffraction data collection For crystals that are of the order of nm in size at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible irrespective of the diffraction technique used Here it is shown that at a very low electron dose at most e- - a Medipix quantum area detector is sufficiently sensitive to allow the collection of a -frame rotation series of keV electron-diffraction data from a single nm thick protein crystal A highly parallel keV electron beam lambda allowed observation of ... More
When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e- �-2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ~100 nm thick protein crystal. A highly parallel 200 keV electron beam ([lambda] = 0.025 �) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4�. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins Less
DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate In eukaryotes DING proteins have been isolated by virtue of their implication in several diseases and biological processes Some of them are potent inhibitors of HIV- replication transcription raising the question of their potential involvement in the human defence system Recently a protein from Pseudomonas aeruginosa strain PA named PA DING or LapC belonging to the DING family has been identified ... More
DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life. They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate. In eukaryotes, DING proteins have been isolated by virtue of their implication in several diseases and biological processes. Some of them are potent inhibitors of HIV-1 replication/transcription, raising the question of their potential involvement in the human defence system. Recently, a protein from Pseudomonas aeruginosa strain PA14, named PA14DING or LapC, belonging to the DING family has been identified. The structure of PA14DING, combined with detailed biochemical characterization and comparative analysis with available DING protein structures, will be helpful in understanding the structural determinants implicated in the inhibition of HIV-�1 by DING proteins. Here, the expression, purification and crystallization of PA14DING and the collection of X-ray data to 1.9 � resolution are reported. Less
In hemophilia A routine prophylaxis with exogenous factor VIII FVIII requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies FVIII inhibitors To overcome these drawbacks we screened asymmetric bispecific IgG antibodies to factor IXa FIXa and factor X FX mimicking the FVIII cofactor function Since the therapeutic potential of the lead bispecific antibody was marginal FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX and the pharmacokinetics was improved by engineering the charge properties of the variable region Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain ... More
In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients. Less
Collapsin response mediator protein- CRMP- is the latest identified member of the CRMP cytosolic phosphoprotein family which is crucial for neuronal development and repair CRMPs exist as homo- and or hetero-tetramers in vivo and participate in signaling transduction cytoskeleton rearrangements and endocytosis CRMP- antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners We determined the crystal structures of a full length and a truncated version of human CRMP- both of which form a homo-tetramer similar to those observed in CRMP- and CRMP- However solution studies indicate that CRMP- and ... More
Collapsin response mediator protein-5 (CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers�in vivo�and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca2+�and Mg2+, destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs. Less
Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of nm were imaged by kV cryo-EM on a Falcon direct electron detector The images were taken close to focus and to the eye appeared devoid of contrast Fourier transforms of the images revealed the reciprocal lattice up to resolution in favourable cases and up to resolution for about half the crystals The reciprocal-lattice spots showed structure indicating that the ordering of the crystals was not uniform Data processing revealed details at higher than resolution and indicated the presence of multiple mosaic blocks within the crystal ... More
Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 � resolution in favourable cases and up to 4 � resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 � resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed. Less
Flavodoxins which exist widely in microorganisms have been found in various pathways with multiple physiological functions The flavodoxin Fld containing the cofactor flavin mononucleotide FMN from sulfur-reducing bacteria Desulfovibrio gigas D gigas is a short-chain enzyme that comprises residues with a molecular mass of kDa and plays important roles in the electron-transfer chain To investigate its structure we purified this Fld directly from anaerobically grown D gigas cells The crystal structure of Fld determined at resolution is a dimer with two FMN packing in an orientation head to head at a distance of which generates a long and connected negatively ... More
Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners. Less
Crystallization of membrane proteins and peptides represents a challenge in the field of structural biology Lipidic cubic phase LCP has become an important medium for crystallogenesis of membrane proteins of different molecular weight Here the small membrane peptide gramicidin is used as an example peptide to test if LCP can produce diffraction quality crystals for membrane proteins and peptides in the lower molecular weight range This approach was initially tested with the standard LCP lipid monoolein and later on extended to a variety of different monoacylglycerols varying in their acyl chain length Data sets for three different crystal forms were ... More
Crystallization of membrane proteins and peptides represents a challenge in the field of structural biology. Lipidic cubic phase (LCP) has become an important medium for crystallogenesis of membrane proteins of different molecular weight. Here, the small membrane peptide gramicidin is used as an example peptide to test if LCP can produce diffraction quality crystals for membrane proteins and peptides in the lower molecular weight range. This approach was initially tested with the standard LCP lipid monoolein and later on extended to a variety of different monoacylglycerols varying in their acyl chain length. Data sets for three different crystal forms were obtained. In each case gramicidin was found in the double stranded double helical (DSDH) conformation. One crystal form shows stabilizing hydrogen bonds between adjacent tryptophan residues indicating how DSDH can be stabilized as an aggregate in the membrane. The cytoplasmic domain of the putative zinc transporter CzrB was solved in the apo and zinc-bound forms. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. Less
The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses which impact on human and animal health Current picornavirus vaccines are frequently produced from inactivated virus Inactivation often reduces the stability of the virus capsid causing a problem for Foot and Mouth Disease Virus FMDV where certain serotypes fall apart into pentameric assemblies below pH or at temperatures slightly above C destroying their effectiveness in eliciting a protective immune response As a result vaccines require a cold chain for storage and animals need to be frequently immunised FMDV is a member of the Aphthovirus genus ... More
The physical properties of viral capsids are major determinants of vaccine efficacy for several
picornaviruses which impact on human and animal health. Current picornavirus vaccines are
frequently produced from inactivated virus. Inactivation often reduces the stability of the
virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain
serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly
above 37�C, destroying their effectiveness in eliciting a protective immune response. As a
result, vaccines require a cold chain for storage and animals need to be frequently immunised.
FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven
FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of
antigenic variation. As part of collaboration between the Division of Structural Biology,
Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South
Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious
copy virus or recombinant empty capsids with improved thermo-stability for improved
vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular
modelling, free energy calculations, X-ray crystallography, electron microscopy and various
biochemical/biophysical techniques were used to design and help characterise the capsids.
For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were
characterised by MD. Promising candidates were then engineered and shown to confer
increased thermo- and pH-stability. Thus, in silico predictions translate into marked
stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method
was used to determine crystal structures for quality assessment and to verify that no
unanticipated structural changes have occurred as a consequence of the modifications made.
The structures of the wildtype and two of the stabilised mutants were solved and the antigenic
surfaces shown to be unchanged.
Animal trials showed stabilised particles can generate a similar or improved neutralising
antibody response compared to the traditional vaccines and may therefore lead to a new
generation of stable and safe vaccines.
Declaration
ii
DECLARATION OF W Less
picornaviruses which impact on human and animal health. Current picornavirus vaccines are
frequently produced from inactivated virus. Inactivation often reduces the stability of the
virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain
serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly
above 37�C, destroying their effectiveness in eliciting a protective immune response. As a
result, vaccines require a cold chain for storage and animals need to be frequently immunised.
FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven
FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of
antigenic variation. As part of collaboration between the Division of Structural Biology,
Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South
Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious
copy virus or recombinant empty capsids with improved thermo-stability for improved
vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular
modelling, free energy calculations, X-ray crystallography, electron microscopy and various
biochemical/biophysical techniques were used to design and help characterise the capsids.
For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were
characterised by MD. Promising candidates were then engineered and shown to confer
increased thermo- and pH-stability. Thus, in silico predictions translate into marked
stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method
was used to determine crystal structures for quality assessment and to verify that no
unanticipated structural changes have occurred as a consequence of the modifications made.
The structures of the wildtype and two of the stabilised mutants were solved and the antigenic
surfaces shown to be unchanged.
Animal trials showed stabilised particles can generate a similar or improved neutralising
antibody response compared to the traditional vaccines and may therefore lead to a new
generation of stable and safe vaccines.
Declaration
ii
DECLARATION OF W Less
The optimum conditions for the formation of plate-like and urchin-like microcrystals of biomolecules and their transfer to rotors for solid-state NMR spectroscopy depend on a variety of factors of which minimizing the manipulation of the microcrystals and storing the sample for several months at K C play an important role Three biological systems were investigated Hen Egg-White HEW lysozyme residues the lengthened C-terminal domain LCter of Human centrin residues and the complex between the C-terminal domain Cter of Human centrin residues and the P -XPC peptide residues
A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described It provides crystals that appear within - min of setup at K The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase which can be daunting as a material when first encountered Having used the protocol to produce crystals of the test protein lysozyme the experimenter can proceed with confidence to apply the method to more valuable membrane and soluble protein targets The ... More
A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described. It provides crystals that appear within 15-30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling of in meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot. Less
The structure of ribose -phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC has been determined at resolution The structure was solved by molecular replacement which identified the functional homodimer in the asymmetric unit Despite only showing sequence identity to its closest homologue the structure adopted the typical a and d- ribose - phosphate isomerase fold Comparison to other related structures revealed high homology in the active site allowing a model of the substrate-bound protein to be proposed The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I - at Diamond Light ... More
The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 � resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical a and � d-�ribose 5-�phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography. Less
Including more than members in humans Rab proteins constitute the largest subfamily of the Ras- like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound inactive and a GTP-bound active conformation The conversion between these states is carried out by regulatory factors GTPase activating proteins GAPs stimulate GTP hydrolysis and guanine nucleotide exchange factors GEFs catalyze the GDP-GTP exchange Rab proteins interact with effector proteins only in the active state thereby regulating vesicular trafficking in eukaryotic cells For this purpose the activity and the intracellular localization of Rab proteins need to be tightly regulated ... More
Including more than 60 members in humans, Rab proteins constitute the largest subfamily of the Ras-
like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound
(inactive) and a GTP-bound (active) conformation. The conversion between these states is carried out by
regulatory factors: GTPase activating proteins (GAPs) stimulate GTP hydrolysis and guanine nucleotide
exchange factors (GEFs) catalyze the GDP-GTP exchange. Rab proteins interact with effector proteins
only in the active state, thereby regulating vesicular trafficking in eukaryotic cells. For this purpose, the
activity and the intracellular localization of Rab proteins need to be tightly regulated. In order to ensure
their own survival, some intracellular pathogens have developed intriguing strategies for manipulation
of intracellular vesicular transport processes and in particular of the Rab proteins involved. A prominent
example of an intracellular pathogen that manipulates Rab proteins for its own benefit is Legionella
pneumophila. In particular, the Legionella protein DrrA (defect in Rab recruitment A) was identified in
the recent past as a protein that manipulates the intracellular localization and activity of Rab1. At the
beginning of this work, structural studies on this protein showed the presence of an additional,
previously uncharacterized domain possessing adenylyltransferase activity towards Rab1. The
characterization of this enzymatic activity was the central subject of this work.
Within this work, the x-ray crystal structure of adenylylated Rab1 was solved. This structure showed that
Rab1 was specifically modified on a tyrosine residue in the functionally important switch II region.
Further studies of the effects of this modification showed that the interaction of Rab1-AMP with GAPs
and the human effector Mical-3 are drastically inhibited, whereas the interaction with the GEF domain
of the Legionella protein DrrA and the Legionella effector protein LidA are not significantly inhibited.
Characterisation of the enzyme kinetics of DrrA and the recently identified deadenylylating enzyme SidD
showed that Rab1:GTP is the preferred substrate of adenylylation by DrrA while SidD possesses a
significantly lower substrate specificity towards the active or inactive conformation of Rab1. This work
includes the first description and characterization of adenylylation as a posttranslational modification of
Rab proteins. In the context of the current literature, the results of this work allowed the proposal of a
model in which adenylylation temporarily inhibits deactivation of Rab1 by GAPs and thus the extraction
of Rab1 from the Legionella containing vacuolar (LCV) membrane by GDI and Rab1 is entrapped at the
LCV membrane. At a later stage of infection, deadenylylation by SidD allows for deactivation and
extraction by GDI. Less
like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound
(inactive) and a GTP-bound (active) conformation. The conversion between these states is carried out by
regulatory factors: GTPase activating proteins (GAPs) stimulate GTP hydrolysis and guanine nucleotide
exchange factors (GEFs) catalyze the GDP-GTP exchange. Rab proteins interact with effector proteins
only in the active state, thereby regulating vesicular trafficking in eukaryotic cells. For this purpose, the
activity and the intracellular localization of Rab proteins need to be tightly regulated. In order to ensure
their own survival, some intracellular pathogens have developed intriguing strategies for manipulation
of intracellular vesicular transport processes and in particular of the Rab proteins involved. A prominent
example of an intracellular pathogen that manipulates Rab proteins for its own benefit is Legionella
pneumophila. In particular, the Legionella protein DrrA (defect in Rab recruitment A) was identified in
the recent past as a protein that manipulates the intracellular localization and activity of Rab1. At the
beginning of this work, structural studies on this protein showed the presence of an additional,
previously uncharacterized domain possessing adenylyltransferase activity towards Rab1. The
characterization of this enzymatic activity was the central subject of this work.
Within this work, the x-ray crystal structure of adenylylated Rab1 was solved. This structure showed that
Rab1 was specifically modified on a tyrosine residue in the functionally important switch II region.
Further studies of the effects of this modification showed that the interaction of Rab1-AMP with GAPs
and the human effector Mical-3 are drastically inhibited, whereas the interaction with the GEF domain
of the Legionella protein DrrA and the Legionella effector protein LidA are not significantly inhibited.
Characterisation of the enzyme kinetics of DrrA and the recently identified deadenylylating enzyme SidD
showed that Rab1:GTP is the preferred substrate of adenylylation by DrrA while SidD possesses a
significantly lower substrate specificity towards the active or inactive conformation of Rab1. This work
includes the first description and characterization of adenylylation as a posttranslational modification of
Rab proteins. In the context of the current literature, the results of this work allowed the proposal of a
model in which adenylylation temporarily inhibits deactivation of Rab1 by GAPs and thus the extraction
of Rab1 from the Legionella containing vacuolar (LCV) membrane by GDI and Rab1 is entrapped at the
LCV membrane. At a later stage of infection, deadenylylation by SidD allows for deactivation and
extraction by GDI. Less
The APPL and APPL proteins APPL adaptor protein phosphotyrosine interaction pleckstrin homology PH domain and leucine zipper-containing protein are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus They play important roles in signal transduction through their ability to act as Rab effectors Rabs are a family of Ras GTPases involved in membrane trafficking Both APPL and APPL comprise an N-terminal membrane-curving BAR Bin-amphiphysin-Rvs domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain The structure and interactions of APPL are well characterized ... More
The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a Kd of 140 nm. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2. Less
Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV- neutralization Here we report an HIV- gp membrane-proximal external region MPER -specific antibody named E which neutralizes of tested viruses An analysis of sera from healthy HIV- -infected donors demonstrated that contained MPER-specific antibodies and contained E -like specificities In contrast to other neutralizing MPER antibodies E did not bind phospholipids was not autoreactive and bound cell-surface envelope The structure of E in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp -hydrophobic residues and a critical Arg Lys ... More
Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ~98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical Arg/Lys just prior to the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 Env. Less
In the recent past macromolecular crystallography has gone through substantial methodological and technological development The purpose of this review is to provide a general overview of structural biology and its impact on enzyme structure function analysis and illustrate how it is modifying the focus of research relevant to alkaloid biosynthesis
PII proteins are central signal processing units for the regulation of nitrogen metabolism in bacteria archaea and plants They act in response to cellular energy carbon and nitrogen availability The central metabolites ATP ADP and -oxoglutarate which indicate cellular energy and carbon nitrogen abundance bind in a highly organized manner to PII and induce effector-molecule-dependent conformational states of the T-loop Depending on these states PII proteins bind and modulate the activity of various regulatory targets A mutant variant of the Synechococcus elongatus PII protein PII-I N has been identified to have impaired - oxoglutarate binding Here the PII-I N variant ... More
PII proteins are central signal processing units for the regulation of nitrogen metabolism in bacteria, archaea and plants. They act in response to cellular energy, carbon and nitrogen availability. The central metabolites ATP, ADP and 2-oxoglutarate, which indicate cellular energy and carbon/nitrogen abundance, bind in a highly organized manner to PII and induce effector-molecule-dependent conformational states of the T-loop. Depending on these states, PII proteins bind and modulate the activity of various regulatory targets. A mutant variant of the Synechococcus elongatus PII protein (PII-I86N) has been identified to have impaired 2-�oxoglutarate binding. Here, the PII-I86N variant was cocrystallized in the presence of ATP, magnesium and citrate and its structure was solved at a resolution of 1.05 �. The PII-I86N variant bound citrate in place of 2-oxoglutarate. Citrate binding is mediated primarily by interactions with the ATP-coordinated magnesium ion and the backbone atoms of the T-�loop. Citrate binding rearranges the conformation of the T-�loop and, consistent with this, citrate suppresses the binding of PII-I86N to an NAG kinase variant, which is similar to the suppression of PII-NAG kinase complex formation by 2-OG. Based on the structures of 2-OG and citrate, homocitrate was suggested as a third ligand and an efficient response towards this molecule with different functional properties was observed. Together, these data provide a first glimpse of a genetically engineered PII variant that senses a new effector molecule. Less
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli purified and crystallized Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen The crystals belonged to the primitive tetragonal space group P with unit-cell parameters a b c Complete data sets were collected from native and selenomethionine-substituted protein crystals at K to and resolution respectively
Structural studies of human G protein-coupled receptors GPCRs have recently been accelerated through the use of the T lysozyme fusion partner that was inserted into the third intracellular loop Using chimeras of the human -adrenergic and human A A adenosine receptors we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs In particular the use of the thermostabilized apocytochrome b RIL as a fusion partner displays certain advantages over the previously utilized T lysozyme resulting in a significant improvement in stability and structure in GPCR-fusion constructs
Members of the opioid receptor family of G-protein-coupled receptors GPCRs are found throughout the peripheral and central nervous system where they have key roles in nociception and analgesia Unlike the classical opioid receptors and -OR -OR and -OR which were delineated by pharmacological criteria in the s and s the nociceptin orphanin FQ N OFQ peptide receptor NOP also known as ORL- was discovered relatively recently by molecular cloning and characterization of an orphan GPCR Although it shares high sequence similarity with classical opioid GPCR subtypes NOP has a markedly distinct pharmacology featuring activation by the endogenous peptide N OFQ ... More
Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the ‘classical’ opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR1. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands2,3. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP–compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands. Less
Second-order nonlinear optical imaging of chiral crystals SONICC is an emerging technique for crystal imaging and characterization We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications Given that they provide near-complete suppression of any background SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis Because SONICC creates high-resolution images nucleation and growth kinetics can also be observed SONICC can detect metastable homochiral crystalline forms of amino acids crystallizing from racemic solutions which confirms ... More
Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald�s rule of stages for crystal growth. SONICC�s selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation. Less
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps This has enabled the crystallization of large numbers of proteins including integral membrane proteins This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality The latter underscores the importance of the ... More
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps. This has enabled the crystallization of large numbers of proteins, including integral membrane proteins. This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success. Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality. The latter underscores the importance of the development of powerful biophysical characterization techniques. Less
As with many other viruses the initial cell attachment of rotaviruses which are the major causative agent of infantile gastroenteritis is mediated by interactions with specific cellular glycans The distally located VP domain of the rotavirus spike protein VP ref mediates such interactions The existing paradigm is that sialidase-sensitive animal rotavirus strains bind to glycans with terminal sialic acid Sia whereas sialidase-insensitive human rotavirus strains bind to glycans with internal Sia such as GM ref Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies it is not yet known how VP of human rotaviruses ... More
As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans1,2,3,4. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies1,3,6,7, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells8, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori9 and noroviruses10. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population. Less
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes including the regulation of pain respiratory drive mood and in the case of -opioid receptor -OR dysphoria and psychotomimesis Here we report the crystal structure of the human -OR in complex with the selective antagonist JDTic arranged in parallel dimers at resolution The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human -OR Modelling of other important -OR-selective ligands including the morphinan-derived antagonists norbinaltorphimine and -guanidinonaltrindole and the diterpene agonist salvinorin A analogue ... More
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. Less
Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input To investigate the conformational changes that relay the regulatory signal we have studied the HAMP domain a ubiquitous intracellular module connecting input to output domains HAMP forms a parallel dimeric four-helical coiled coil and rational substitutions in our model domain Af HAMP induce a transition in its interhelical packing characterized by axial rotation of all four helices the gearbox signaling model We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af HAMP variants to the DHp domain of EnvZ a ... More
Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity. Less
Cytoskeletal intermediate filaments IFs assemble from the elementary dimers based on a segmented -helical coiled-coil CC structure Crystallographic studies of IF protein fragments remain the main route to access their atomic structure To enable crystallization such fragments must be sufficiently short As a consequence they often fail to assemble into the correct CC dimers In particular human vimentin fragment D corresponding to the first half of coil residues stays monomeric in solution We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus The crystal structure ... More
Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261–335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3 Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed. Less
A broad working definition of structural proteomics SP is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules Recently the process for protein structure determination has become highly automated and SP platforms have been established around the globe utilizing X-ray crystallography as a tool Although protein structures often provide clues about the biological function of a target once the three-dimensional structures have been determined bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles This article reviews the current status of SP methods for the structure determination pipeline ... More
A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation. Less