1181 Citations
RNA binding proteins RBPs are involved in many cellular functions To facilitate functional characterization of RBPs we generated an RNA interference RNAi library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability we screened the library using a total ATP assay and high-throughput imaging in Drosophila S R cultured cells The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays Altogether we provide resources in the ... More
RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online. Less
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases which shape and remodel membranes in diverse cellular processes Dynamin forms predominantly tetramers in the cytosol which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane Previous studies have described the architecture of dynamin dimers but the molecular determinants for dynamin assembly and its regulation have remained unclear Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state Combining structural data with mutational studies oligomerization measurements and Markov state models of molecular dynamics simulations ... More
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes1. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane1. Previous studies have described the architecture of dynamin dimers2,3, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot–Marie–Tooth neuropathy4 and centronuclear myopathy5, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction. Less
Organophosphorus OP pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics such as tacrine are characterized by reversible binding Historically the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because similarly to humans guinea pigs have low amounts of circulating OP metabolizing carboxylesterase To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development guinea pig acetylcholinesterase gpAChE was cloned into ... More
Organophosphorus (OP) pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics, such as tacrine, are characterized by reversible binding. Historically, the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because, similarly to humans, guinea pigs have low amounts of circulating OP metabolizing carboxylesterase. To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development, guinea pig acetylcholinesterase (gpAChE) was cloned into pENTR/D-TOPO, recombined into pT-Rex-DEST30 and expressed in Human Embryonic Kidney 293 cells. Recombinant gpAChE was purified to a specific activity of 800 U/mg using size exclusion and immobilized nickel affinity chromatography, with purity confirmed by gel electrophoresis. Ellman’s assay was used to enzymatically characterize gpAChE, identifying a KM of 154±18.7 µmol L-1 and a kcat of 4.79x104±5.26x102 /sec. Apparent gpAChE IC50’s for diisopropylfluorophosphate, dicrotophos, paraoxon, and an Alzheimer’s drug, tacrine, were found to be 10.1±1.98, 337±108, 1.02±0.29 and 0.30±0.01 µmol L-1, respectively. Apparent gpAChE inhibition constants for diisopropylfluorophosphate, dicrotophos, paraoxon, and tacrine were found to be 8.40±0.60, 4.50±0.30, 0.29±0.01 and 0.42±0.07 µmol L-1, respectively. Lineweaver-Burk plots confirmed tacrine as a mixed inhibitor and paraoxon, dicrotophos and diisopropylfluorophosphate as irreversible non-competitive inhibitors. gpAChE bimolecular rate constants for diisopropylfluorophosphate, dicrotophos and paraoxon were found to be 1.44±0.33x104, 1.56±0.12x103 and 4.57± 0.23x105 L µmol-1 min-1, respectively. Although the blood levels of OP metabolizing carboxylesterases in the guinea pig are similar to the low levels in human blood, the gpAChE is different in its enzymology. Therefore, medical countermeasures against OP intoxication should be tested for efficacy with the recombinant form of gpAChE prior to initiating animal studies. Less
PRMT is a protein arginine methyltransferase involved in transcriptional regulation human immunodeficiency virus pathogenesis DNA base excision repair and cell cycle progression Like other PRMTs PRMT is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target In the present study we described six crystal structures of PRMT from Mus musculus solved and refined at for the highest resolution structure The crystal structures revealed that the folding of the helix X is required to stabilize a productive active site before methylation of the bound peptide can occur In the absence of cofactor metal cations can ... More
PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7. Less
Crystallization of biological macromolecules such as proteins implies several prerequisites for example the presence of one or more initial nuclei sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process The initiation of a crystallization process itself is a stochastic event forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable However early identification of the occurrences of unit cells is the key to nanocrystal detection The optical ... More
Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy. Less
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules that ensure cellular homeostasis Structure determination of those complexes in a purified state is often a tedious undertaking due to the compositional complexity and the related relative structural instability To improve the stability of macromolecular complexes in vitro we present here a generic method that optimizes the stability homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behaviour in the presence of various buffers and small molecules The method includes the automated analysis of thermal unfolding curves based on a newly developed biophysical unfolding model for ... More
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules that ensure cellular homeostasis. Structure determination of those complexes in a purified state is often a tedious undertaking due to the compositional complexity and the related relative structural instability. To improve the stability of macromolecular complexes in vitro, we present here a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behaviour in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a newly developed biophysical unfolding model for complexes. We found that under stabilizing conditions even large multi-component complexes reveal an almost ideal two-state unfolding behaviour. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and Cryo EM. Less
The potentially structured core domain of the intrinsically disordered protein Knr from Saccharomyces cerevisiae comprising residues was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method Selenomethionine-containing SeMet protein was also purified and crystallized Crystals of both proteins belonged to space group P with unit-cell parameters a b c for the native protein and a b c for the SeMet protein and diffracted to and resolution respectively There are two molecules in the asymmetric unit related by a twofold axis The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the ... More
The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80–340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6522, with unit-cell parameters a = b = 112.44, c = 265.21 Å for the native protein and a = b = 112.49, c = 262.21 Å for the SeMet protein, and diffracted to 3.50 and 3.60 Å resolution, respectively. There are two molecules in the asymmetric unit related by a twofold axis. The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the identification of secondary-structure elements. Less
-Xylosidases EC catalyze the hydrolysis of short xylooligosaccharides into xylose which is an essential step in the complete depolymerization of xylan the major hemicellulosic polysaccharide of plant cell walls and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry In this study a GH -xylosidase identified from the bacterium Bacillus licheniformis BlXylA was cloned into the the pET- a bacterial expression vector recombinantly overexpressed in Escherichia coli BL DE cells and purified to homogeneity by metal-affinity and size-exclusion chromatography The protein was crystallized in the presence of the organic solvent -methyl- -pentanediol and a single ... More
β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family. Less
G protein-coupled receptors GPCRs signal primarily through G proteins or arrestins Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin determined by serial femtosecond X-ray laser crystallography Together with extensive biochemical and mutagenesis data the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements including TM and Helix to recruit arrestin Correspondingly arrestin adopts the pre-activated conformation with a rotation between the ... More
G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. Less
Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins nucleic acids and viruses Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics However X-ray crystallography still has a few fundamental limitations some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology
The present study was undertaken to evaluate the anti-influenza vasorelaxant and vasoprotective potential of a series of flavan- -ols and proanthocyanidins and to gain insight into the mode of action The first part of this thesis addressed the inhibitory potential of the tested substances against a bacterial V cholera and a viral influenza A California H N neuraminidase NA using a MuNANA -fluorescence-based assay The present study verified that the substrate MuNANA is recognized by both NAs Compared to the positive controls zanamivir und oseltamivir carboxylic acid the studied proanthocyanidin enriched fractions Salix spp Nelia meyeri Cephalophyllum spp Betula spp ... More
The present study was undertaken to evaluate the anti-influenza, vasorelaxant and vasoprotective potential of a series of flavan-3-ols and proanthocyanidins and to gain insight into the mode of action. The first part of this thesis addressed the inhibitory potential of the tested substances against a bacterial (V. cholera) and a viral influenza A (California/04/2009[H1N1]) neuraminidase (NA), using a (MuNANA)-fluorescence-based assay. The present study verified that the substrate MuNANA is recognized by both NAs. Compared to the positive controls zanamivir und oseltamivir carboxylic acid, the studied proanthocyanidin enriched fractions (Salix spp., Nelia meyeri, Cephalophyllum spp., Betula spp., Potentilla erecta, Rhus leptodictya, Diospyros kaki, Pelargonium sidoides) and ellagitannins (Geraniin, Granatin A, Carpinusin, Terchebin, Casuariin, Vescalagin, Paeonianin C) were significantly more effective against the bacterial NA. In contrast, all polyphenolic samples were far less effective inhibitors of the viral NA than the reference samples. Structure-activity relationships indicated that the degree of polymerization, the 2,3-stereochemistry, hydroxylation patterns and the presence of a 3-O-galloyl group largely affected the inhibitory potential of the tested flavan-3-ols and proanthocyanidins. The degree of galloylation appeared crucial in the series of ellagitannins. In addition, the combination of Zanamivir with EPs® 7630 (1:10) showed a synergistic inhibitory effect in studies against V. cholerae NA. The evaluation of co-crystal data of the bacterial respectively viral NA and either Zanamivir or oseltamivir carboxylate showed due to the interactions with the only viral occurring 150-loop both drugs inhibit the viral enzyme more effectively than the bacterial NA. In the second part of this thesis the vasorelaxant potential was investigated using a tissue bath protocol. Porcine coronary arterial rings were contracted with U46619 and relaxed by cumulative addition of the polyphenolic samples. Within the series of flavan-3-ols and proanthocyanidin- enriched fractions, galloylated compounds induced a concentration-dependent vasodilatation. The experiments showed that Diospyros-proanthocyanidins and procyanidin B2-3-O-gallate stimulated phosphoinositide-3-kinase/protein kinase B, endothelial NO synthase, and soluble guanylyl cyclase, respectively. Both samples evoked an endothelium-dependent relaxations via the NO/cGMP pathway. In addition, Na+/K+-ATPase was involved in the relaxant response to these polyphenols. Intracellular ROS formation in response to the proanthocyanidin samples was verified and the mitochondrial respiratory chain identified as a potential source of reactive oxygen species for the procyanidin B2-3-O-gallate. Less
The second-harmonic generation SHG activity of protein crystals was found to be enhanced by up to -fold by the intercalation of SHG phores within the crystal lattice Unlike the intercalation of fluorophores the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice In addition the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response Kinetics studies indicate that intercalation arises over a timeframe of several minutes ... More
The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ~1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy. Less
Suppressor of cytokine signalling SOCS is the substrate-binding component of a Cullin-RING E ubiquitin ligase CRL complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system As a key regulator of the transcriptional response to growth signals SOCS and its protein complex partners are potential targets for small molecule development We found that crystals of SOCS in complex with its adaptor proteins Elongin C and Elongin B underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments To solve the phase problem for the new crystal form we identified the presence of arsenic atoms ... More
Suppressor of cytokine signalling 2 (SOCS2) is the substrate-binding component of a Cullin-RING E3 ubiquitin ligase (CRL) complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system. As a key regulator of the transcriptional response to growth signals, SOCS2 and its protein complex partners are potential targets for small molecule development. We found that crystals of SOCS2 in complex with its adaptor proteins, Elongin C and Elongin B, underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments. To solve the phase problem for the new crystal form we identified the presence of arsenic atoms in the crystals, a result of covalent modification of cysteines by cacodylate, and successfully extracted anomalous signal from these atoms for experimental phasing. The resulting structure provides a means for solving future structures where the crystals must be treated with DMSO for ligand soaking approaches. Additionally, the conformational changes induced in this structure reveal flexibility within SOCS2 that match those postulated by previous molecular dynamics simulations. This conformational flexibility illustrates how SOCS2 can orient its substrates for successful ubiquitination by other elements of the CRL complex. Less
Rhodopsin is a membrane protein from the G protein-coupled receptor family Together with its ligand retinal it forms the visual pigment responsible for night vision In order to perform ultrafast dynamics studies a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin In such an approach microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser XFEL after a precise photoactivation delay Here a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols i screening the low-salt crystallization conditions preferred for ... More
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup. Less
In response to adenosine -diphosphate the P Y receptor P Y R facilitates platelet aggregation and thus serves as an important antithrombotic drug target Here we report the crystal structures of the human P Y R in complex with a nucleotide antagonist MRS at resolution and with a non-nucleotide antagonist BPTU at resolution The structures reveal two distinct ligand binding sites providing atomic details of P Y R s unique ligand binding modes MRS recognizes a binding site within the seven transmembrane bundle of P Y R which however is different in shape and location from the nucleotide binding site ... More
In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. Less
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology Bacteriorhodopsin from Halobium salinarum bR is a striking example of the difficulties in membrane protein overexpression We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression This complementary protein approach allowed us to increase bR expression by two orders of magnitude ... More
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. Less
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism However it has not been studied as extensively as other biological agents and very few of its proteins have been structurally characterized To address this situation we undertook a study of critical metabolic enzymes in C burnetii that have great potential as drug targets We used high-throughput techniques to produce novel crystal structures of of these proteins We selected one protein C burnetii dihydrofolate reductase CbDHFR for additional work to demonstrate the value of these structures for structure-based drug design This enzyme's structure reveals a feature in the ... More
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in�C. burnetii�that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein,�C. burnetii�dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by�in silico�screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria. Less
Chlorophyll is one of the most abundant pigments worldwide Every year chlorophyll is not only newly synthesized but big amounts of chlorophyll are degraded during fruit ripening and in senescing leaves The pathway of chlorophyll breakdown the so called PAO phyllobilin pathway has been extensively studied over the past decades Most of the involved genes have been cloned and characterized except for the activity responsible for magnesium removal of chlorophyll and some of the side chain-modifying enzymes Pheophytinase PPH is one of the first enzymes involved in chlorophyll degradation specifically hydrolyzing the ester bond between the porphyrin ring and the ... More
Chlorophyll is one of the most abundant pigments worldwide. Every year, chlorophyll is not only newly synthesized, but big amounts of chlorophyll are degraded during fruit ripening and in senescing leaves. The pathway of chlorophyll breakdown, the so called PAO/phyllobilin pathway, has been extensively studied over the past decades. Most of the involved genes have been cloned and characterized, except for the activity responsible for magnesium removal of chlorophyll and some of the side chain-modifying enzymes. Pheophytinase (PPH) is one of the first enzymes involved in chlorophyll degradation, specifically hydrolyzing the ester bond between the porphyrin ring and the phytol moiety of pheophytin, but not of chlorophyll. In this work, the substrate specificity of PPH was further determined. PPH can be characterized as an esterase with tight specificity for the acid moiety, which is the porphyrin ring. The KM for pheophytin a and for two other accepted substrates with similar molecular structure is in the µM range, indicating high substrate affinities. Chlorophyll is a likely inhibitor of the enzymatic activity. To reveal the substrate-binding mechanism of PPH, a crystallization approach was performed. To this end, one focus of this work was to elucidate a good purification system for PPH. However, purification and tag-separation turned out to be difficult for PPH and first potential crystallization conditions were defined with the uncleaved PPH-MBP fusion. In this work I also demonstrate that PPH is not only involved in leaf senescence, but also in chlorophyll degradation during fruit ripening. For a comparison study Tomato was chosen as a model plant. I could show that tomato PPH is an ortholog of Arabidopsis thaliana PPH and is responsible for phytol cleavage in senescing tomato leaves. The PAO/ phyllobilin pathway is active in ripening fruits and PPH activity was found in chromoplasts. However, the absence of PPH did not impair color break in fruits, indicating that other, so far unknown, hydrolases are active in parallel. A last focus of this work was on the identification of factors responsible for magnesium dechelation from chlorophyll, the first step in the breakdown process. I obtained a first good indication that this is a non-enzymatic process, where changes in the local pH trigger the loss of magnesium. However, additional experiments will be required to corroborate this hypothesis. Less
Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain PD that promotes virulence by detecting the macrophage phagosome PhoQ activity is repressed by divalent cations and induced in environments of acidic pH limited divalent cations and cationic antimicrobial peptides CAMP Previously it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in a-helices and suggesting this region contributes to pH sensing Therefore we engineered a disulfide bond between W C and A C in the ... More
Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in a-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in a-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence. Less
Nisin is a -amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics Nisin displays a high bactericidal activity against various Gram-positive bacteria including some human-pathogenic strains However there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome The encoded protein NSR cleaves off the last six amino acids of nisin thereby reducing its bactericidal efficacy An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops resulting ... More
Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. Less
Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions A crystal volume optimization method is described and associated with a phase diagram for ... More
Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization. Less
The lipid cubic phase LCP continues to grow in popularity as a medium in which to generate crystals of membrane and soluble proteins for high-resolution X-ray crystallographic structure determination To date the PDB includes records attributed to the LCP or in meso method Among the listings are some of the highest profile membrane proteins including the -adrenoreceptor Gs protein complex that figured in the award of the Nobel Prize in Chemistry to Lefkowitz and Kobilka The most successful in meso protocol to date uses glass sandwich crystallization plates Despite their many advantages glass plates are challenging to harvest crystals from ... More
The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the �2-adrenoreceptor�Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 � using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development. Less
We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems However there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners Here we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail We have implemented this screen on several macromolecular complexes from a variety of organisms revealing novel profiles even for well-studied proteins Our approach is robust economical ... More
We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. Less
Upon binding to its bacterial host receptor the tail tip of phage T perforates by an unknown mechanism the heavily armoured cell wall of the host This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions In the perspective of a structural study of the phage tail we have systematically overproduced eight of the eleven T tail proteins with or without a N- or a C-terminal His -tag The widely used Hi -tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography The presence of a ... More
Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. Less
Pmi an enzyme of unknown function from Proteus Mirabilis HI and the amidohydrolase superfamily was cloned purified to homogeneity and functionally characterized The three-dimensional structure of Pmi was determined with zinc and cacodylate bound in the active site PDB id RHG The structure was also determined with manganese and butyrate in the active site PDB id QSF Pmi folds as a distorted a -barrel that is typical for members of the amidohydrolase superfamily and cog The substrate profile for Pmi was determined via a strategy that marshaled the utilization of bioinformatics structural characterization and focused library screening The protein was ... More
Pmi1525, an enzyme of unknown function from Proteus Mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). The structure was also determined with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (�/a)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. The substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat/Km values of 580 s-1 and 1.2 � 105 M-1 s-1, respectively) and 4-nitrophenyl butyrate (kcat and kcat/Km values of 140 s-1 and 1.4 � 105 M-1 s-1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents. Less
In this study we take advantage of a recently described chimera of the a nicotinic acetylcholine receptor nAChR and acetylcholine binding protein AChBP termed a -AChBP To date more than crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site Here we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in a -AChBP Using electrophysiological recordings on the human a nAChR we demonstrate that each of the three sites is involved in ... More
In this study we take advantage of a recently described chimera of the a7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed a7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in a7-AChBP. Using electrophysiological recordings on the human a7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels. Less
HIV- -neutralizing antibodies develop in most HIV- -infected individuals although highly effective antibodies are generally observed only after years of chronic infection Here we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC through longitudinal sampling of peripheral B cell transcripts over years and co-crystal structures of lineage members Next-generation sequencing identified VRC -lineage transcripts which encompassed diverse antibodies organized into distinct phylogenetic clades Prevalent clades maintained characteristic features of antigen recognition though each evolved binding loops and disulfides that formed distinct recognition surfaces Over the course of the study ... More
HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization. Less
Angiotensin II type receptor AT R is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance Although several anti-hypertensive drugs have been developed as AT R blockers ARBs the structural basis for AT R ligand-binding and regulation has remained elusive mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser we successfully determined the room-temperature crystal structure of the human AT R in complex with its selective antagonist ZD at resolution The AT R-ZD ... More
Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 � resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. Less
POTs proton-dependent oligopeptide transporters are integral membrane proteins and essential for maintaining homeostasis in cells by switching between two major conformations during the transport cycle Di- and tripeptides as well as some small peptide like compounds are recognized and transported across the membrane This fact leads to the pharmacological interest in these transporters for drug delivery In humans two transporters PepT and PepT occur So far only structures of five bacterial homologues are available Eukaryotic POTs exist of transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices It was possible to clone different prokaryotic transporter constructs into pET expression ... More
POTs (proton-dependent oligopeptide transporters) are integral membrane proteins and essential for maintaining homeostasis in cells by switching between two major conformations during the transport cycle. Di- and tripeptides as well as some small peptide like compounds are recognized and transported across the membrane. This fact leads to
the pharmacological interest in these transporters for drug delivery. In humans two transporters, PepT1 and PepT2, occur. So far only structures of five bacterial homologues are available. Eukaryotic POTs exist of 12 transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices. It was possible to clone different prokaryotic transporter constructs into pET expression vectors as well as the PepT-like transporter (CtPOT) of Chaetomium thermophilum, a thermophilic eukaryote, and the human PepT2. Two transporters of Shewanella oneidensis (PepTSo2) modified with the thermostabilized apocytochrome b562RIL as well as the CtPOT and PepT2 were expressed in Escherichia coli cells. These transporters except PepT2 were solubilized and purified. Crystals were obtained of all proteins but it was not possible to solve any structure so far due to limited crystal diffraction. Less
the pharmacological interest in these transporters for drug delivery. In humans two transporters, PepT1 and PepT2, occur. So far only structures of five bacterial homologues are available. Eukaryotic POTs exist of 12 transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices. It was possible to clone different prokaryotic transporter constructs into pET expression vectors as well as the PepT-like transporter (CtPOT) of Chaetomium thermophilum, a thermophilic eukaryote, and the human PepT2. Two transporters of Shewanella oneidensis (PepTSo2) modified with the thermostabilized apocytochrome b562RIL as well as the CtPOT and PepT2 were expressed in Escherichia coli cells. These transporters except PepT2 were solubilized and purified. Crystals were obtained of all proteins but it was not possible to solve any structure so far due to limited crystal diffraction. Less
Synthesis of '- '-oligoadenylates - A by oligoadenylate synthetase OAS is an important innate cellular response that limits viral replication by activating the latent cellular RNase RNase L to degrade single-stranded RNA Some rotaviruses and coronaviruses antagonize the OAS RNase L pathway through the activity of an encoded H phosphoesterase domain that cleaves - A These viral H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein AKAP and share a core structure and an active site that contains two well-defined HF S T F where F is a hydrophobic residue motifs but their mechanism of substrate binding ... More
Synthesis of 2'-5'-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HF(S/T)F (where F is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave �-sheet that contains the catalytic cleft, but it lacks two a-helical regions and two �-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. Less
Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices The technique is as simple as drawing with the reagent pencils on a device When aqueous samples are added to the device the reagents dissolve from the pencil matrix and become available to react with analytes in the sample Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution Most importantly sensitive reagents such as enzymes are stable in the ... More
Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment. Less
Recently the first known light-driven sodium pumps from the microbial rhodopsin family were discovered We have solved the structure of one of them Krokinobacter eikastus rhodopsin KR in the monomeric blue state and in two pentameric red states at resolutions of and and respectively The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface that the ion-release cavity is capped by a unique N-terminal -helix and that the ion-uptake cavity is unexpectedly large and open to the surface Obstruction of the cavity with the mutation G F imparts KR ... More
Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics. Less
Crystallization of integral membrane proteins MPs is notoriously difficult given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process MP crystallization in a membrane mimetic matrix known as lipidic cubic phase LCP has recently started to gain popularity following successes in structure determination of G protein-coupled receptors GPCRs transporters and enzymes Unlike crystallization trials in aqueous solutions where protein molecules are free to move diffusion of MPs in LCP is restricted and thus a high level of protein mobility can serve as an early indication for subsequent crystallization success Prompted by ... More
Crystallization of integral membrane proteins (MPs) is notoriously difficult, given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process. MP crystallization in a membrane mimetic matrix, known as lipidic cubic phase (LCP), has recently started to gain popularity, following successes in structure determination of G protein-coupled receptors (GPCRs), transporters, and enzymes. Unlike crystallization trials in aqueous solutions where protein molecules are free to move, diffusion of MPs in LCP is restricted, and, thus, a high level of protein mobility can serve as an early indication for subsequent crystallization success. Prompted by our initial observations that precipitant conditions can dramatically affect diffusion of GPCRs in LCP, we have developed a simple precrystallization assay, based on measuring protein diffusion at a number of different conditions by fluorescence recovery after photobleaching (LCP-FRAP). Over the last few years, the LCP-FRAP assay was incorporated in our GPCR structure determination pipeline and proved as a powerful technique allowing for a faster identification of crystallization conditions for many different receptors. The assay is used to screen for the best protein constructs, ligands, LCP host lipids, precipitants, and additives, thereby focusing subsequent crystallization trials on the most promising parts of the multidimensional crystallization phase diagram, substantially increasing the likelihood of finding the right crystallization condition. Here, we describe our LCP-FRAP protocols for guiding GPCR crystallization, which can be adapted to any other MP, and discuss some of the critical considerations related to application of this assay. Less
The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas is the basis of genome editing applications in numerous model organisms and cell types In a binary complex with a dual crRNA tracrRNA guide or single-molecule guide RNA Cas targets double-stranded DNAs harboring sequences complementary to a -nucleotide segment in the guide RNA Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition Here we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas that allowed us to reconstitute and crystallize the enzyme ... More
The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes. Less
Na -translocating NADH quinone oxidoreductase NQR is a redox-driven sodium pump operating in the respiratory chain of various bacteria including pathogenic species The enzyme has a unique set of redox active prosthetic groups which includes two covalently bound flavin mononucleotide FMN residues attached to threonine residues in subunits NqrB and NqrC The reason of FMN covalent bonding in the subunits has not been established yet In the current work binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding To study ... More
Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. Less
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase QRS an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA MARS complex is therefore lacking Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma ... More
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural–functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. Less
Bi-functional - and - opioid receptor OR ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects We solved the structure of human -OR bound to the bi-functional -OR antagonist and -OR agonist tetrapeptide H-Dmt -Tic -Phe -Phe -NH DIPP-NH by serial femtosecond crystallography revealing a cis-peptide bond between H-Dmt and Tic The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides and to develop improved analgesics
Antibodies are complex macromolecules and their phase behavior as well as interactions within different solvents and precipitants are still not understood To shed some light into the processes on a molecular dimension the occurring self-interactions between antibody molecules were analyzed by means of the osmotic second virial coefficient B The determined B follows qualitatively the phenomenological Hofmeister series describing the aggregation probability of antibodies for the various solvent compositions However a direct correlation between crystallization probability and B in form of a crystallization slot does not seem to be feasible for antibodies since the phase behavior is strongly dependent on ... More
Antibodies are complex macromolecules and their phase behavior as well as interactions within different solvents and precipitants are still not understood. To shed some light into the processes on a molecular dimension, the occurring self-interactions between antibody molecules were analyzed by means of the osmotic second virial coefficient (B22). The determined B22 follows qualitatively the phenomenological Hofmeister series describing the aggregation probability of antibodies for the various solvent compositions. However, a direct correlation between crystallization probability and B22 in form of a crystallization slot does not seem to be feasible for antibodies since the phase behavior is strongly dependent on their anisotropy. Kinetic parameters have to be taken into account due to the molecular size and complexity of the molecules. This is confirmed by a comparison of experimental data with a theoretical phase diagram. On the other hand the solubility is thermodynamically driven and therefore the B22 could be used to establish a universal solubility line for the monoclonal antibody mAb04c and different solvent compositions by using thermodynamic models. � 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:438�451, 2015 Less
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry Proteins can either be soluble crystalline or precipitated Additionally liquid liquid phase separation gelation and skin formation can occur A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed For lysozyme from chicken egg white human lysozyme glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH and Sodium chloride ammonium sulfate polyethylene glycol and polyethylene glycol were used as precipitants Crystallizing conditions could be found for lysozyme from chicken ... More
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid–liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution. Less
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