888 Citations
Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP which are universal second messengers Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain CNBD of these channels is thought to cause a conformational change that promotes channel opening The C-linker domain which connects the channel pore to this CNBD plays an important role in coupling ligand binding to channel opening Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker CNBD from hyperpolarization-activated cyclic nucleotide-modulated HCN channels However these structures reveal little to no conformational changes ... More
Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels. Less
Xyloglucan-specific endo- - -glucanases Xegs EC exhibit high catalytic specificity for - linkages of xyloglucan a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species In nature GH Xegs are not associated with carbohydrate-binding modules CBMs and here we have investigated the effect of the fusion of the xyloglucan-specific CBM on the structure and function of a GH Xeg from Aspergillus niveus XegA This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA An increased k cat chimera s - XegA s - and reduced KM chimera mg mL - ... More
Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera. Less
The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallization trials required However the pH is usually recorded as that of the buffer solution which can be highly inaccurate
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data The traditional method of data reduction is very simple results are summarized in peer-reviewed publications which are hopefully published in high-impact journals By their nature publications include only the most important results derived from experiments that may have been performed over the course of many years The main content of the published paper is a concise compilation of these data an interpretation of the experimental results and a comparison of these results with those obtained by other scientists Due to an avalanche of structural biology manuscripts submitted to ... More
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data. The traditional method of data reduction is very simple�results are summarized in peer-reviewed publications, which are hopefully published in high-impact journals. By their nature, publications include only the most important results derived from experiments that may have been performed over the course of many years. The main content of the published paper is a concise compilation of these data, an interpretation of the experimental results, and a comparison of these results with those obtained by other scientists.
Due to an avalanche of structural biology manuscripts submitted to scientific journals, in many recent cases descriptions of experimental methodology (and sometimes even experimental results) are pushed to supplementary materials that are only published online and sometimes may not be reviewed as thoroughly as the main body of a manuscript. Trouble may arise when experimental results are contradicting the results obtained by other scientists, which requires (in the best case) the reexamination of the original raw data or independent repetition of the experiment according to the published description of the experiment. There are reports that a significant fraction of experiments obtained in academic laboratories cannot be repeated in an industrial environment (Begley CG & Ellis LM, Nature 483(7391):531�3, 2012). This is not an indication of scientific fraud but rather reflects the inadequate description of experiments performed on different equipment and on biological samples that were produced with disparate methods. For that reason the goal of a modern data management system is not only the simple replacement of the laboratory notebook by an electronic one but also the creation of a sophisticated, internally consistent, scalable data management system that will combine data obtained by a variety of experiments performed by various individuals on diverse equipment. All data should be stored in a core database that can be used by custom applications to prepare internal reports, statistics, and perform other functions that are specific to the research that is pursued in a particular laboratory.
This chapter presents a general overview of the methods of data management and analysis used by structural genomics (SG) programs. In addition to a review of the existing literature on the subject, also presented is experience in the development of two SG data management systems, UniTrack and LabDB. The description is targeted to a general audience, as some technical details have been (or will be) published elsewhere. The focus is on �data management,� meaning the process of gathering, organizing, and storing data, but also briefly discussed is �data mining,� the process of analysis ideally leading to an understanding of the data. In other words, data mining is the conversion of data into information. Clearly, effective data management is a precondition for any useful data mining. If done properly, gathering details on millions of experiments on thousands of proteins and making them publicly available for analysis�even after the projects themselves have ended�may turn out to be one of the most important benefits of SG programs. Less
Due to an avalanche of structural biology manuscripts submitted to scientific journals, in many recent cases descriptions of experimental methodology (and sometimes even experimental results) are pushed to supplementary materials that are only published online and sometimes may not be reviewed as thoroughly as the main body of a manuscript. Trouble may arise when experimental results are contradicting the results obtained by other scientists, which requires (in the best case) the reexamination of the original raw data or independent repetition of the experiment according to the published description of the experiment. There are reports that a significant fraction of experiments obtained in academic laboratories cannot be repeated in an industrial environment (Begley CG & Ellis LM, Nature 483(7391):531�3, 2012). This is not an indication of scientific fraud but rather reflects the inadequate description of experiments performed on different equipment and on biological samples that were produced with disparate methods. For that reason the goal of a modern data management system is not only the simple replacement of the laboratory notebook by an electronic one but also the creation of a sophisticated, internally consistent, scalable data management system that will combine data obtained by a variety of experiments performed by various individuals on diverse equipment. All data should be stored in a core database that can be used by custom applications to prepare internal reports, statistics, and perform other functions that are specific to the research that is pursued in a particular laboratory.
This chapter presents a general overview of the methods of data management and analysis used by structural genomics (SG) programs. In addition to a review of the existing literature on the subject, also presented is experience in the development of two SG data management systems, UniTrack and LabDB. The description is targeted to a general audience, as some technical details have been (or will be) published elsewhere. The focus is on �data management,� meaning the process of gathering, organizing, and storing data, but also briefly discussed is �data mining,� the process of analysis ideally leading to an understanding of the data. In other words, data mining is the conversion of data into information. Clearly, effective data management is a precondition for any useful data mining. If done properly, gathering details on millions of experiments on thousands of proteins and making them publicly available for analysis�even after the projects themselves have ended�may turn out to be one of the most important benefits of SG programs. Less
The catalytic domain of the trimeric human -enoyl-CoA isomerase type HsECI has the typical crotonase fold In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the E- or Z-enoyl-CoA substrate molecules A catalytic glutamate is essential for the proton transfer between the substrate C and C atoms for forming the product E-enoyl-CoA which is a key intermediate in the -oxidation pathway The active site is covered by the C-terminal helix- In HsECI the isomerase domain is extended at its N terminus by an acyl-CoA binding protein ACBP ... More
The catalytic domain of the trimeric human ?3,?2-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the �-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis. Less
The modified base -formylcytosine fC was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase Genomics and proteomics studies have suggested an additional role for fC in transcription regulation through chromatin remodeling Here we propose that fC might affect these processes through its effect on DNA conformation Biophysical and structural analysis revealed that fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those ... More
The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase. Genomics and proteomics studies have suggested an additional role for 5fC in transcription regulation through chromatin remodeling. Here we propose that 5fC might affect these processes through its effect on DNA conformation. Biophysical and structural analysis revealed that 5fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those comprising other cytosine modifications. The 1.4-�-resolution X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shows how 5fC changes the geometry of the grooves and base pairs associated with the modified base, leading to helical underwinding. Less
For the successful application of protein crystallization as a downstream step a profound knowledge of protein phase behavior in solutions is needed Therefore a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol PEG First the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional -D phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing In comparison to the use of salts as precipitant PEG was more suitable to obtain compact -D crystals over a ... More
For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account. Less
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps Here we present the crystal structure of a new member of the family Haloarcula marismortui bacteriorhodopsin I HmBRI D N mutant at the resolution of While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps its proton release region is extended and contains additional water molecules The protein's fold is reinforced by three novel inter-helical hydrogen bonds two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins Despite the expression in ... More
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies. Less
Wax esters WEs are esters of fatty acids and fatty alcohols They can cover a broad range of physical properties which makes them especially interesting for industrial applications including additives in cosmetics and high class lubricants Biosynthesis of WEs is a widespread feature in nature and is carried out by two essential classes of enzymes One of them is the class of fatty acyl reductases FARs which reduce acyl-CoAs or acyl-acyl carrier proteins acyl-ACPs to the corresponding fatty alcohols The second one is the class of wax synthases WSs which esterify fatty alcohols with acyl CoAs yielding WEs The majority ... More
Wax esters (WEs) are esters of fatty acids and fatty alcohols. They can cover a broad range of physical properties, which makes them especially interesting for industrial applications, including additives in cosmetics and high class lubricants. Biosynthesis of WEs is a widespread feature in nature and is carried out by two essential classes of enzymes. One of them is the class of fatty acyl reductases (FARs), which reduce acyl-CoAs or acyl-acyl carrier proteins (acyl-ACPs) to the corresponding fatty alcohols. The second one is the class of wax synthases (WSs), which esterify fatty alcohols with acyl CoAs, yielding WEs. The majority of FARs and WSs described to date exhibit a broad substrate range, resulting in wax blends of heterogeneous compositions. With respect to a commercial production of WEs in genetically modified plants, a defined WE blend is desired rather than a mixture of WEs. Enzymes with improved substrate specificities, tailored for the production of individual WE blends, are one way to overcome these issues. However, the required knowledge about structure-function relationships in FARs and WSs for the construction of respective enzymes is lacking to date. The aim of the present thesis was thus to elucidate structural determinants of substrate specificity in FARs and WSs. To date, crystal structures are neither available from FARs nor from WSs. Hence, four FARs (DmFAR1 from Drosophila melanogaster, MaFAR1 from Marinobacter aquaeolei, MmFAR1 from Mus musculus and TcFAR1 from Tribolium castaneum), two WSs (AbWSD1 from Acinetobacter baylyi and MmAWAT2 from Mus musculus) and a soluble diacylglycerol O-acyltransferase (AtDGAT3 from Arabidopsis thaliana) were studied in order to obtain structural insights. The activity of DmFAR1 was first described in the present work. It produces tetracosanol and hexacosanol upon expression in yeast, while expression in E. coli yielded tetradecanol, hexadecanol, hexadecenol and octadecenol. DmFAR1, MaFAR1, MmFAR1, AbWSD1, MmAWAT2 and AtDGAT3 were expressed in and purified from E. coli. Although this resulted in aggregated proteins in most cases, AbWSD1 was obtained in a quaternary structure corresponding to a trimer, when expressed as a fusion protein with the bacterial trigger factor. The respective fusion protein reproducibly formed crystals, which diffracted to 2.1 Å. Attempts to soak the crystals with iodine led to disruption of the crystals. Hence, the respective selenoprotein was produced and applied to crystallisation screens, which were ongoing at the end of this thesis. In order to elucidate substrate specificity determining structures in MmAWAT2, comparative studies of MmAWAT2 and MmDGAT2, which share ~ 70 % homology, were carried out. Both enzymes are acyltransferases and are capable of synthesising both, TAGs and WEs. Furthermore, both enzymes show distinct substrate specificities with respect to WE synthesis. Two predicted, neighboured hairpin forming transmembrane (TM) domains were identified to have an influence on the substrate specificity of MmAWAT2. Chimeric enzyme variants of MmAWAT2 carrying the respective section of the MmDGAT2 sequence showed a severely altered acyl chain incorporation pattern into WEs as compared to both, MmAWAT2 and MmDGAT2. Furthermore, respective variants showed an altered ratio of produced WEs and TAGs. This phenotype was also exhibited by the MmAWAT2 single amino acid exchange variant N36R, carrying a mutation in the part of the sequence which encodes the two predicted TM domains. Thus, the predicted TM domains of MmAWAT2 seem to have a role in substrate specificity determination of the enzyme. This work provides further insights into structure function relationships concerning substrate specificity in DGAT2-type acyltransferases. Furthermore, the successful crystallisation of a WS might pave the way for an extensive comprehension of this class of enzymes. Less
Hydrocarbon stapling can restore bioactive -helical structure to natural peptides yielding research tools and prototype therapeutics to dissect and target protein interactions Here we explore the capacity of peptide stapling to generate high-fidelity protease-resistant mimics of antigenic structures for vaccine development HIV- has been refractory to vaccine technologies thus far although select human antibodies can broadly neutralize HIV- by targeting sequences of the gp juxtamembrane fusion apparatus To develop candidate HIV- immunogens we generated and characterized stabilized -helices of the membrane-proximal external region SAH-MPER of gp SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing E and ... More
Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination. Less
The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma resulting in worldwide morbidity in dogs S pseudintermedius also causes life-threatening human infections Furthermore methicillin-resistant S pseudintermedius is emerging resembling the human health threat of methicillin-resistant Staphylococcus aureus Therefore it is increasingly important to characterize targets for intervention strategies to counteract S pseudintermedius infections Here we used biophysical methods mutagenesis and X-ray crystallography to define the ligand-binding properties and structure of SitA an S pseudintermedius surface lipoprotein SitA was strongly and specifically stabilized by Mn and Zn ions Crystal structures of SitA complexed with Mn and Zn ... More
The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma, resulting in worldwide morbidity in dogs. S. pseudintermedius also causes life-threatening human infections. Furthermore, methicillin-resistant S. pseudintermedius is emerging, resembling the human health threat of methicillin-resistant Staphylococcus aureus. Therefore it is increasingly important to characterize targets for intervention strategies to counteract S. pseudintermedius infections. Here we used biophysical methods, mutagenesis, and X-ray crystallography, to define the ligand-binding properties and structure of SitA, an S. pseudintermedius surface lipoprotein. SitA was strongly and specifically stabilized by Mn2+ and Zn2+ ions. Crystal structures of SitA complexed with Mn2+ and Zn2+ revealed a canonical class III solute-binding protein with the metal cation bound in a cavity between N- and C-terminal lobes. Unexpectedly, one crystal contained both apo- and holo-forms of SitA, revealing a large side-chain reorientation of His64, and associated structural differences accompanying ligand binding. Such conformational changes may regulate fruitful engagement of the cognate ABC (ATP-binding cassette) transporter system (SitBC) required for metal uptake. These results provide the first detailed characterization and mechanistic insights for a potential therapeutic target of the major canine pathogen S. pseudintermedius, and also shed light on homologous structures in related staphylococcal pathogens afflicting humans. Less
Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell generating functional or aberrant states The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle including -strand to -helix and -helix to -strand transitions These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane
Structural and functional studies require the development of sophisticated Big Data technologies and software to increase the knowledge derived and ensure reproducibility of the data This paper presents summaries of the Structural Biology Knowledge Base the VIPERdb Virus Structure Database evaluation of homology modeling by the Protein Model Portal the ProSMART tool for conformation-independent structure comparison the LabDB super laboratory information management system and the Cambridge Structural Database These techniques and technologies represent important tools for the transformation of crystallographic data into knowledge and information in an effort to address the problem of non-reproducibility of experimental results
Amphipols APols have become important tools for the stabilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs solubilized in APols would be of high importance for structural biology However despite considerable efforts it is still not clear whether MP APol complexes can form well-ordered crystals suitable for X-ray crystallography In the present work we show that an APol-trapped MP can be crystallized in meso Bacteriorhodopsin BR trapped by APol A - was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The crystals diffract beyond The structure of ... More
Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs. Less
The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallo graphy Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes here a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals thereby prioritizing for visual inspection those images that are most likely to contain useful information The use of textons is introduced to describe crystallization droplets objectively allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence ... More
The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallo�graphy. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes; here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate; additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals. Less
The inner membrane ring of the bacterial type III secretion system TTSS is composed of two proteins In Chlamydia trachomatis this ring is formed by CdsD gene name CT and CdsJ gene name CTA CdsD consists of amino acids The last amino acids at its C-terminal end relate it to the type III secretion system YscD HrpQ protein family The C-terminal domain consisting of amino acids of C trachomatis CdsD was overexpressed in Escherichia coli and purified using immobilized metal-affinity chromatography IMAC and size-exclusion chromatography The protein was crystallized using the vapour-diffusion method A data set was collected to resolution ... More
The inner membrane ring of the bacterial type III secretion system (TTSS) is composed of two proteins. In Chlamydia trachomatis this ring is formed by CdsD (gene name CT_664) and CdsJ (gene name CTA_0609). CdsD consists of 829 amino acids. The last 400 amino acids at its C-terminal end relate it to the type III secretion system YscD/HrpQ protein family. The C-terminal domain, consisting of amino acids 558–771, of C. trachomatis CdsD was overexpressed in Escherichia coli and purified using immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. The protein was crystallized using the vapour-diffusion method. A data set was collected to 2.26 Å resolution. The crystals have the symmetry of space group C2, with unit-cell parameters a = 106.60, b = 23.91, c = 118.65 Å, β = 104.95°. According to the data analysis there is expected to be one molecule in the asymmetric unit, with a Matthews coefficient of 3.0 Å3 Da−1. Less
Rotavirus RV nonstructural protein NSP is a virulence factor that disrupts cellular Ca homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea Although its native oligomeric state is unclear crystallographic studies of the coiled-coil domain CCD of NSP from two different strains suggest that it functions as a tetramer or a pentamer While the CCD of simian strain SA NSP forms a tetramer that binds Ca at its core the CCD of human strain ST forms a pentamer lacking the bound Ca despite the residues E and Q that coordinate Ca binding being conserved In ... More
Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca2+ homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca2+ at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca2+ despite the residues (E120 and Q123) that coordinate Ca2+ binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca2+ binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca2+ or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca2+ at neutral pH and forms a tetramer, it does not bind Ca2+ at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca2+ binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca2+ may form a basis for its pleiotropic functions during RV replication.
IMPORTANCE The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca2+-bound tetrameric state and a Ca2+-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca2+-bound tetramer to a Ca2+-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4. Less
IMPORTANCE The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca2+-bound tetrameric state and a Ca2+-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca2+-bound tetramer to a Ca2+-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4. Less
The isolation of human monoclonal antibodies mAbs is providing important insights regarding the specificities that underlie broad neutralization of HIV- reviewed in Here we report a broad and extremely potent HIV-specific mAb termed O which binds novel HIV- envelope glycoprotein Env epitope O neutralized of pseudoviruses with an IC g ml The median IC of neutralized viruses was g ml among the most potent thus far described O did not bind monomeric forms of Env tested but did bind the trimeric BG SOSIP Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it ... More
The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 �g/ml. The median IC50 of neutralized viruses was 0.033 �g/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design. Less
Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality Seeding is one such optimization method In classical seeding experiments the seed crystals are put into new albeit similar conditions The past decade has seen the emergence of an alternative seeding strategy microseed matrix screening MMS In this strategy the seed crystals are transferred into conditions unrelated to the seed source Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups better diffracting crystals and crystallization of previously uncrystallizable targets MMS can be ... More
Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems. Less
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase LCP-SFX for protein structure determination at X-ray free electron lasers XFELs LCP-SFX uses the gel-like lipidic cubic phase LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection LCP is a liquid-crystalline mesophase composed of lipids and water It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room ... More
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase (LCP-SFX) for protein structure determination at X-ray free electron lasers (XFELs). LCP-SFX uses the gel-like lipidic cubic phase (LCP) as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase, composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room temperature high-resolution data with minimal effects of radiation damage from sub-10 �m crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared to SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2�3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3�5 days, including the time required for crystals to grow. Less
The LabDB laboratory information management system LIMS tracks organizes and analyzes data from chemical and solution management protein production crystallization diffraction structure solution and in vitro biochemical and biophysical experiments The system is comprised of multiple modules specialized for different tasks such as the Xtaldb system for crystallization or the hkldb module of the HKL- suite for diffraction data collection and structure solution The biochemical biophysical experiments tracked by LabDB include spectrophotometric binding and kinetics thermal shift binding isothermal titration calorimetry ITC and protein quantitation These tools associate functional and structural experiments for example for selecting likely substrates for co-crystallization ... More
The LabDB laboratory information management system (LIMS) tracks, organizes and analyzes data from chemical and solution management, protein production, crystallization, diffraction, structure solution, and in vitro biochemical and biophysical experiments. The system is comprised of multiple modules specialized for different tasks, such as the Xtaldb system for crystallization or the hkldb module of the HKL-3000 suite for diffraction data collection and structure solution. The biochemical/biophysical experiments tracked by LabDB include spectrophotometric binding and kinetics, thermal shift binding, isothermal titration calorimetry (ITC) and protein quantitation. These tools associate functional and structural experiments, for example, for selecting likely substrates for co-crystallization and soaking experiments. Whenever possible, the system harvests data with no or minimal user intervention from laboratory hardware. Devices that may connect to or import data into LabDB include crystal observation (Rigaku Minstrel HT and Formulatrix Rock Imager), liquid handling (Formulatrix Rock Maker and Emerald Opti-Matrix Maker), chromatography (GE Healthcare AKTA), quantitation (Caliper LabChip GX II and Bio-Rad Gel Doc EZ), RT-PCR (Applied Biosystems 7900HT and Bio-Rad C1000/CFX96) and ITC (MicroCal iTC-200) systems. LabDB is used by two high-throughput PSI:Biology centers (MCSG and NYSGRC) as well as other major NIH consortia (the Center for Structural Genomics of Infectious Diseases and the Enzyme Function Initiative), and track millions of experiments on tens of thousands of targets.[1] The system also provides extensive data mining and analysis tools for translating raw experimental data into information and knowledge. We present examples of analyses generated by the system useful in designing new experiments. Less
Protein crystallization with microseed matrix screening: application to human germline antibody Fabs
The crystallization of human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening MMS In initial screening diffraction-quality crystals were obtained for only three Fabs while many Fabs produced hits that required optimization Application of MMS using the initial screens and or refinement screens resulted in diffraction-quality crystals of these Fabs Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization The crystallization protocols and strategies that resulted in structure determination of all Fabs are ... More
The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization. Less
Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins Single influences on the protein are not understood and fundamental knowledge remains to be obtained For this purpose a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams This approach was applied to three monoclonal antibodies to investigate influences of pH protein and salt concentrations with five different salts being tested Although differences exist between the antibodies this extensive study confirmed the general applicability ... More
Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. � 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1103�1113, 2014 Less
The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens Here we characterize PmDsbA from Proteus mirabilis a bacterial pathogen increasingly associated with multidrug resistance PmDsbA exhibits the characteristic properties of a DsbA including an oxidizing potential destabilizing disulfide acidic active site cysteine and dithiol oxidase catalytic activity We evaluated a peptide PWATCDS derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA The crystal structures of PmDsbA and the ... More
The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. Less
The Smoothened receptor SMO mediates signal transduction in the hedgehog pathway which is implicated in normal development and carcinogenesis SMO antagonists can suppress the growth of some tumors however mutations at SMO have been found to abolish their anti-tumor effects a phenomenon known as chemoresistance Here we report three crystal structures of human SMO bound to the antagonists SANT and Anta XV and the agonist SAG at resolution The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites where SANT binds at a deeper site as compared with other ligands Distinct interactions at D ... More
The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors; however, mutations at SMO have been found to abolish their anti-tumor effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6–2.8Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D4736.55 elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules. Less
A systematic study of the crystallization of an a-helical integral membrane enzyme diacylglycerol kinase DgkA using the lipidic cubic mesophase or in meso method is described These trials have resulted in the production of blocky rhombohedron-shaped crystals of diffraction quality currently in use for structure determination Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied These monoacylglycerol lipids incorporated fatty acyl chains ranging from to carbon atoms long with cis olefinic bonds located toward the middle of ... More
A systematic study of the crystallization of an a-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 �C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials. Less
Metabolic pathways in eubacteria and archaea often are encoded by operons and or gene clusters genome neighborhoods that provide important clues for assignment of both enzyme functions and metabolic pathways We describe a bioinformatic approach genome neighborhood network GNN that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions metabolic pathways of uncharacterized enzymes in protein families We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily PRS InterPro IPR The predictions were verified by measuring in vitro activities for proteins in ... More
Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes. Less
Lipidic cubic phase LCP is a gel-like liquid crystalline membrane-mimetic matrix It has been successfully used to stabilize and crystallize challenging membrane proteins such as G protein-coupled receptors the structure of which is often difficult to obtain by other methods Despite many advantages the LCP crystallization method has not been widely adopted because of difficulties associated with handling highly viscous LCP material Recent advances in the development of tools and instruments for LCP crystallization are aimed at facilitating the research in this area and to help structural biologists in integrating these technologies in their working routine
Phospholipids have major roles in the structure and function of all cell membranes Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain IPCT fused with a membrane CDP-alcohol phosphotransferase domain DIPPS at resolution The bifunctional protein dimerizes through the DIPPS domains each comprising six transmembrane -helices The active site cavity is hydrophilic and widely ... More
Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed. Less
Type-A -aminobutyric acid receptors GABAARs are the principal mediators of rapid inhibitory synaptic transmission in the human brain A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia anxiety and epilepsy Here we present the first three-dimensional structure of a GABAAR the human homopentamer at resolution This structure reveals architectural elements unique to eukaryotic Cys-loop receptors explains the mechanistic consequences of multiple human disease mutations and shows a surprising structural role for a conserved N-linked glycan The receptor was crystallised bound to a previously unknown agonist benzamidine opening a new avenue for the rational design of GABAAR modulators ... More
Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows a surprising structural role for a conserved N-linked glycan. The receptor was crystallised bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitised state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission. Less
Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles which transduce signals generated in an N-terminal sensor domain across nm to a conserved C-terminal signaling subdomain This signal transduction regulates the activity of associated kinases altering the behavior of the flagellar motor and hence cell motility Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain We have determined the structure of a chimeric protein consisting of the HAMP domain from Archaeoglobus fulgidus Af and the methyl-accepting domain of Escherichia coli Tsr It shows a nm coiled coil that alternates between ... More
Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15–20 nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21 nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation–demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors. Less
Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA Automation of the individual steps of a crystallization experiment from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments has been the response to address this issue Today large high-throughput crystallization facilities many of them open to the general user community are capable of setting up thousands of crystallization trials per day It is thus possible to test multiple constructs of each target for their ability to form crystals on a ... More
Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given. Less
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane Here we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations We show that after ligand release the apo transport domain adopts a compact and occluded conformation that can traverse the membrane completing the transport cycle Sodium binding primes the transport domain to accept its substrate ... More
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues. Less
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function Consequently functions are assigned to the vast majority of proteins via automated homology-based methods with the result that as many as are incorrectly annotated or unannotated Schnoes et al PLoS Comput Biol e PMC free article PubMed Google Scholar This manuscript describes a study of the d-mannonate dehydratase ManD subgroup of the enolase superfamily ENS to investigate how function diverges as sequence diverges Previously one member of the subgroup had ... More
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605 [PMC free article] [PubMed] [Google Scholar]). This manuscript describes a study of the d-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of d-mannonate to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized to sample sequence�function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 103 to 104 M�1 s�1) for dehydration of d-mannonate, (2) low efficiency (kcat/KM = 101 to 102 M�1 s�1) for dehydration of d-mannonate and/or d-gluconate, and 3) no-activity with either d-mannonate or d-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes d-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004, 576, 133�136 [PubMed] [Google Scholar]) (Ahmed et al. Biochem. J. 2005, 390, 529�540 [PMC free article] [PubMed] [Google Scholar]). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs. Less
The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity Despite its success the method is in its infancy as far as rational crystallogenesis is concerned Consequently significant time effort and resources are still required to generate structure-grade crystals especially with a new target type Therefore a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types Recently a strategy for crystallizing a prokaryotic a-helical membrane protein diacylglycerol kinase DgkA by the in meso method was reported Cryst Growth Des ... More
The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic a-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des. 2013, 13, 2846-2857 [PMC free article] [PubMed] [Google Scholar]). Here, we describe its application to the human a-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 �C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method. Less
PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure In cyanobacteria PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling and these interactions are modulated by ADP ATP and -oxoglutarate These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure Here we used the PII protein from Synechococcus elongatus PCC to reveal the structural basis of anti-cooperative ADP binding Furthermore we clarified the mutual influence of PII-receptor interaction and sensing of the ATP ADP ratio The crystal structures of two ... More
PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the PII-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction. Less
Human noroviruses NoVs cause acute epidemic gastroenteritis Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens HBGAs which are also critical for NoV attachment to host cells Human NoVs are classified into two major genogroups genogroup I GI and GII with each genogroup further divided into several genotypes GII NoVs are more prevalent and exhibit periodic emergence of new variants suggested to be driven by altered HBGA binding specificities and antigenic drift Recent epidemiological studies show increased activity among GI NoVs with some members showing the ability to bind nonsecretor HBGAs ... More
Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. Human NoVs are classified into two major genogroups (genogroup I [GI] and GII), with each genogroup further divided into several genotypes. GII NoVs are more prevalent and exhibit periodic emergence of new variants, suggested to be driven by altered HBGA binding specificities and antigenic drift. Recent epidemiological studies show increased activity among GI NoVs, with some members showing the ability to bind nonsecretor HBGAs. NoVs bind HBGAs through the protruding (P) domain of the major capsid protein VP1. GI NoVs, similar to GII, exhibit significant sequence variations in the P domain; it is unclear how these variations affect HBGA binding specificities. To understand the determinants of possible strain-specific HBGA binding among GI NoVs, we determined the structure of the P domain of a GI.7 clinical isolate and compared it to the previously determined P domain structures of GI.1 and GI.2 strains. Our crystallographic studies revealed significant structural differences, particularly in the loop regions of the GI.7 P domain, altering its surface topography and electrostatic landscape and potentially indicating antigenic variation. The GI.7 strain bound to H- and A-type, Lewis secretor, and Lewis nonsecretor families of HBGAs, allowing us to further elucidate the structural determinants of nonsecretor HBGA binding among GI NoVs and to infer several contrasting and generalizable features of HBGA binding in the GI NoVs. Less
In the Gram-negative enterobacterium Erwinia Pectobacterium and Serratia sp ATCC intrinsic resistance to the carbapenem antibiotic -carbapen- -em- -carboxylic acid is mediated by the CarF and CarG proteins by an unknown mechanism Here we report a high-resolution crystal structure for the Serratia sp ATCC carbapenem resistance protein CarG This structure of CarG is the first in the carbapenem intrinsic resistance CIR family of resistance proteins from carbapenem-producing bacteria The crystal structure shows the protein to form a homodimer in agreement with results from analytical gel filtration The structure of CarG does not show homology with any known antibiotic resistance proteins ... More
In the Gram-negative enterobacterium Erwinia (Pectobacterium) and Serratia sp. ATCC 39006, intrinsic resistance to the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid is mediated by the CarF and CarG proteins, by an unknown mechanism. Here, we report a high-resolution crystal structure for the Serratia sp. ATCC 39006 carbapenem resistance protein CarG. This structure of CarG is the first in the carbapenem intrinsic resistance (CIR) family of resistance proteins from carbapenem-producing bacteria. The crystal structure shows the protein to form a homodimer, in agreement with results from analytical gel filtration. The structure of CarG does not show homology with any known antibiotic resistance proteins nor does it belong to any well-characterised protein structural family. However, it is a close structural homologue of the bacterial inhibitor of invertebrate lysozyme, PliI-Ah, with some interesting structural variations, including the absence of the catalytic site responsible for lysozyme inhibition. Both proteins show a unique �-sandwich fold with short terminal a-helices. The core of the protein is formed by stacked anti-parallel sheets that are individually very similar in the two proteins but differ in their packing interface, causing the splaying of the two sheets in CarG. Furthermore, a conserved cation binding site identified in CarG is absent from the homologue. Less
The vertebrate sodium Nav channel is composed of an ion-conducting a subunit and associated subunits Here we report the crystal structure of the human subunit immunoglobulin Ig domain a functionally important component of Nav channels in neurons and cardiomyocytes Surprisingly we found that the subunit Ig domain assembles as a trimer in the crystal asymmetric unit Analytical ultracentrifugation confirmed the presence of Ig domain monomers dimers and trimers in free solution and atomic force microscopy imaging also detected full-length subunit monomers dimers and trimers Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers ... More
The vertebrate sodium (Nav) channel is composed of an ion-conducting a subunit and associated � subunits. Here, we report the crystal structure of the human �3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the �3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length �3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length �3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that �3 subunits can bind to more than one site on the Nav 1.5 a subunit and induce the formation of a subunit oligomers, including trimers. Our results suggest a new and unexpected role for the �3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations. Less
Lipidic cubic phase LCP crystallization has proven successful for high-resolution structure determination of challenging membrane proteins Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals providing a continuously renewed source of material for serial femtosecond crystallography Data collected from sub- - m-sized crystals produced with less than mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor
Antibodies m and F are the only effective human HIV- -neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region MPER of the gp subunit of the HIV- envelope glycoprotein Although F has been extensively characterized much less is known about antibody m or antibody m a closely related light-chain variant Here we report the crystal structure of m in complex with its gp epitope along with unbound structures of m and m We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp and determined the molecular basis that ... More
Antibodies m66.6 and 2F5 are the only effective human HIV-1-neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein. Although 2F5 has been extensively characterized, much less is known about antibody m66.6 or antibody m66, a closely related light-chain variant. Here, we report the crystal structure of m66 in complex with its gp41 epitope, along with unbound structures of m66 and m66.6. We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp41 and determined the molecular basis that underlies their neutralization of HIV-1. When bound by m66, the N-terminal region of the gp41 MPER adopts a conformation comprising a helix, followed by an extended loop. Comparison of gp41-bound m66 to unbound m66.6 identified three light-chain residues of m66.6 that were confirmed through mutagenesis to underlie the greater breadth of m66.6-mediated virus neutralization. Recognition of gp41 by m66 also revealed similarities to antibody 2F5 both in the conformation of crucial epitope residues as well as in the angle of antibody approach. Aromatic residues at the tip of the m66.6 heavy-chain third complementarity-determining region, as in the case of 2F5, were determined to be critical for virus neutralization in a manner that correlated with antibody recognition of the MPER in a lipid context. Antibodies m66, m66.6, and 2F5 thus utilize similar mechanistic elements to recognize a common gp41-MPER epitope and to neutralize HIV-1. Less
The multidrug resistance-encoding IncA C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria A plasmid-encoded disulfide isomerase is associated with conjugation Sequence analysis of several IncA C plasmids and IncA C-related integrative and conjugative elements ICE from commensal and pathogenic bacteria identified a conserved DsbC DsbG homolog DsbP The crystal structure of DsbP reveals an N-terminal domain a linker region and a C-terminal catalytic domain A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs Overall the structure and redox properties of ... More
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer. Less
Opioids represent widely prescribed and abused medications although their signal transduction mechanisms are not well understood Here we present the high-resolution crystal structure of the human -opioid receptor -OR revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity The distinctive -OR sodium ion site architecture is centrally located in a polar interaction network in the -transmembrane bundle core with the sodium ion stabilizing a reduced agonist affinity state and thereby modulating signal transduction Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn to alanine ... More
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn131 to alanine or valine augments constitutive arrestin-ergic signaling. Asp95Ala, Asn310Ala, and Asn314Ala mutations transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealing that sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor. Less
Protein arginine methyltransferase PRMT is a unique but less characterized member of the family of protein arginine methyltransferases PRMTs that plays a role in male germline gene imprinting PRMT is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem PRMT genes from five different species were cloned and expressed in Escherichia coli and Sf insect cells Four gave soluble proteins from Sf cells of which two were homogeneous and one gave crystals The mouse PRMT structure was solved by the single anomalous dispersion method ... More
Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 � resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 � and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. Less
The ryanodine receptor RyR is a heterotetrameric Ca release channel located on the sarcoplasmic reticulum SR membrane of different cell types RyR type RyR is the dominant isoform in skeletal muscle and RyR type RyR is abundant in the heart The RyR N-terminus is a large cytoplasmic domain that binds many channel modulators including the immunophilin calstabin Calstabins FKBPs which are cis-trans peptidyl-prolyl isomerases modify and bind to RyRs Calstabin FKBP is associated with RyR and calstabin FKBP binds to RyR The binding site for calstabins on RyRs has been studied and includes a proline The proline is preceded by ... More
The ryanodine receptor (RyR) is a heterotetrameric Ca2+ release channel located
on the sarcoplasmic reticulum (SR) membrane of different cell types. RyR type 1 (RyR1)
is the dominant isoform in skeletal muscle and RyR type 2 (RyR2) is abundant in the
heart. The RyR N-terminus is a large cytoplasmic domain that binds many channel
modulators, including the immunophilin calstabin.
Calstabins (FKBPs) which are cis-trans peptidyl-prolyl isomerases modify
and bind to RyRs. Calstabin1 (FKBP12) is associated with RyR1 and calstabin2
(FKBP12.6) binds to RyR2. The binding site for calstabins on RyRs has been studied
and includes a proline. The proline is preceded by a valine or an isoleucine in both RyR
isoforms. Calstabins bind to the immunosuppressive drugs rapamycin and FK506; this
binding suppresses the isomerase activity of these drugs. It has been proposed that this
inhibition is caused by the ability of the immunosuppressive compounds to mimic the
transition state of ligand isomerization.
RyR undergoes several types of post-translational modifications. One of these
modifications, phosphorylation by protein kinase A (PKA) at Ser2808, causes a decrease
in affinity of calstabin to the channel. The dissociation of calstabin from the channel
increases channel openings and promotes sub-conductance states. This phenomenon
causes Ca2+ �leak� from the SR into the cytoplasm and depletes the Ca2+ stores of the cell.
The aberrant release of Ca2+
can promote different disease states. For example, SR Ca
2+
leak in cardiac cells can promote heart failure (HF) and fatal ventricular arrhythmias.
The Marks lab demonstrated that a calstabin2 mutant � in which Asp37 was
mutated into valine � retained the ability to bind to PKA-phosphorylated channels.
Single channel measurements have shown that binding of the calstabin2-D37V restored
the calstabin2-bound channel properties.
In the present study we aimed to structurally understand the differences in binding
between wt-calstabin2 and D37V-calstabin2. To this end, we cloned, expressed and
purified the D37V-calstabin2 with an MBP fusion protein. The fusion protein was
crystallized in the presence of rapamycin and the structure was solved using molecular
replacement techniques. The main difference between the mutant and wt calstabin2 was
that a hydrogen bond between D37 and rapamycin was replaced with a van der Waals
interaction.
We also docked the mutant calstabin2-D37V into our cryo-EM structure of RyR1.
We were able to clearly see that the amino acids D (or V) interacted with a helix
projecting from the RyR structure, which we believe to contain the proline previously
identified by the Marks group. Calstabin2 interacted with the receptor via three distinct
domains; this interaction has implications for coupled gating, phosphorylation and
disease-associated mutations.
The binding affinity of the wt and mutant calstabins was measured using
radiolabeled versions of wt and D37V proteins. We found that the affinity of wt
calstabin2 to PKA-phosphorylated RyR2 decreased threefold compared to non-
phosphorylated RyR. The D37V mutant, however, was able to bind to both
phosphorylated and non-phosphorylated RyR2 with the same affinity.
This study also included efforts to crystallize different RyR fragments. We
attempted to crystallize RyR1 and RyR2 domains that are involved in RyR regulation by
small modulators or domains that are important to its activity. Despite not being able to
crystallize these fragments, we present our results here and suggest they could serve us in
the future for a variety of biochemical and biophysical studies Less
on the sarcoplasmic reticulum (SR) membrane of different cell types. RyR type 1 (RyR1)
is the dominant isoform in skeletal muscle and RyR type 2 (RyR2) is abundant in the
heart. The RyR N-terminus is a large cytoplasmic domain that binds many channel
modulators, including the immunophilin calstabin.
Calstabins (FKBPs) which are cis-trans peptidyl-prolyl isomerases modify
and bind to RyRs. Calstabin1 (FKBP12) is associated with RyR1 and calstabin2
(FKBP12.6) binds to RyR2. The binding site for calstabins on RyRs has been studied
and includes a proline. The proline is preceded by a valine or an isoleucine in both RyR
isoforms. Calstabins bind to the immunosuppressive drugs rapamycin and FK506; this
binding suppresses the isomerase activity of these drugs. It has been proposed that this
inhibition is caused by the ability of the immunosuppressive compounds to mimic the
transition state of ligand isomerization.
RyR undergoes several types of post-translational modifications. One of these
modifications, phosphorylation by protein kinase A (PKA) at Ser2808, causes a decrease
in affinity of calstabin to the channel. The dissociation of calstabin from the channel
increases channel openings and promotes sub-conductance states. This phenomenon
causes Ca2+ �leak� from the SR into the cytoplasm and depletes the Ca2+ stores of the cell.
The aberrant release of Ca2+
can promote different disease states. For example, SR Ca
2+
leak in cardiac cells can promote heart failure (HF) and fatal ventricular arrhythmias.
The Marks lab demonstrated that a calstabin2 mutant � in which Asp37 was
mutated into valine � retained the ability to bind to PKA-phosphorylated channels.
Single channel measurements have shown that binding of the calstabin2-D37V restored
the calstabin2-bound channel properties.
In the present study we aimed to structurally understand the differences in binding
between wt-calstabin2 and D37V-calstabin2. To this end, we cloned, expressed and
purified the D37V-calstabin2 with an MBP fusion protein. The fusion protein was
crystallized in the presence of rapamycin and the structure was solved using molecular
replacement techniques. The main difference between the mutant and wt calstabin2 was
that a hydrogen bond between D37 and rapamycin was replaced with a van der Waals
interaction.
We also docked the mutant calstabin2-D37V into our cryo-EM structure of RyR1.
We were able to clearly see that the amino acids D (or V) interacted with a helix
projecting from the RyR structure, which we believe to contain the proline previously
identified by the Marks group. Calstabin2 interacted with the receptor via three distinct
domains; this interaction has implications for coupled gating, phosphorylation and
disease-associated mutations.
The binding affinity of the wt and mutant calstabins was measured using
radiolabeled versions of wt and D37V proteins. We found that the affinity of wt
calstabin2 to PKA-phosphorylated RyR2 decreased threefold compared to non-
phosphorylated RyR. The D37V mutant, however, was able to bind to both
phosphorylated and non-phosphorylated RyR2 with the same affinity.
This study also included efforts to crystallize different RyR fragments. We
attempted to crystallize RyR1 and RyR2 domains that are involved in RyR regulation by
small modulators or domains that are important to its activity. Despite not being able to
crystallize these fragments, we present our results here and suggest they could serve us in
the future for a variety of biochemical and biophysical studies Less
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data In chapter several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process The new MULTICOMB program discussed in chapter provides a new phase combination algorithm for the density modification step of the structure solution process MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction SAD data directly accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment Testing of ... More
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition. Less
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
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
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
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