Open-access and free articles in Acta Crystallographica Section F: Structural Biology Communications
Acta Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structural genomics initiatives or from iterative studies such as those used in the pharmaceutical industry are particularly welcomed. Articles are available online when ready, making publication as fast as possible, and include unlimited free colour illustrations, movies and other enhancements. The editorial process is completely electronic with respect to deposition, submission, refereeing and publication.
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Open-access and free articles in Acta Crystallographica Section F Structural Biology Communications
Acta Crystallographica Section F Structural Biology Communications
Crystal structure of a tRNA acceptor-stem mimic at 1.94 Å resolution
Despite the theoretical advantages of phosphorus single-wavelength anomalous diffraction (P-SAD) for nucleic acid phasing, its application remains limited due to high atomic displacement parameters and an unfavourable ratio of unique reflections to anomalous scatterers. In this study, we report the crystal structure of an RNA complex composed of four strands, which was solved by experimental phasing after AlphaFold3 failed to produce reliable models. Bromine single-wavelength anomalous diffraction (Br-SAD) data were collected at 0.916 Å on beamline I04 at Diamond Light Source, while phosphorus anomalous data were obtained at 3.024 Å on beamline I23. The structure was successfully phased using bromine anomalous scattering, and phosphorus anomalous peaks corroborated the backbone positions and validated the model. Attempts to phase the structure directly from phosphorus data failed, consistent with theoretical predictions that successful SAD phasing requires a significantly higher reflection-to-scatterer ratio. The final models reveal an RNA complex stabilized by Watson–Crick and Hoogsteen base pairing, forming a pseudo-helical complex instead of the anticipated hairpin stem-loop, likely reflecting crystallization artefacts. This work demonstrates the complementary use of bromine and phosphorus anomalous signals in RNA crystallography.
CRYSTALLOGRAPHY; ANOMALOUS SCATTERING; NUCLEIC ACIDS; TRNA ACCEPTOR STEM; RNA TRIPLEX; PREBIOTIC RNA
Acta Crystallographica Section F: Structural Biology Communications
Sample purification and characterization of the α- and β-crustacyanin pigments from the American lobster for crystallographic and cryo-EM studies
The striking blue hue of live Homarus americanus, known as the American lobster, arises from the interaction of astaxanthin with the carotenoprotein α-crustacyanin. The mechanism underlying the bathochromic shift of the chromophore from its unbound red form (λmax = 472 nm) to the blue protein-bound form (λmax = 631 nm) is the subject of attention from the food and nutraceutical industries for the development of versatile food colourants. Here, we present sample purification and characterization of the α- and β-crustacyanin pigments from the American lobster for crystallographic studies and cryo-EM. Moving from H. gammarus to H. americanus for complex isolation, together with an integrated biophysical characterization, resulted in the production, for the first time, of α-crustacyanin crystals that diffracted to 6.3 Å resolution at 〈I/σ(I)〉 = 1.0 and high-quality negative-stain and cryo-EM images.
ASTAXANTHIN; CRUSTACYANIN; ABSORPTION; CRYSTALLOGRAPHY; CRYO-EM; CAROTENOIDS
Potential 14-3-3 binding sites in sirtuins reveal extended phosphosite-recognition modes
The adapter proteins of the 14-3-3 family modulate the activity and/or localization of their binding partners, which they capture if a generic target motif is in its phosphorylated state. Here, we report the identification of potential 14-3-3 binding sites in human sirtuin deacylases by bioinformatic analysis. We then characterize the interactions of peptides representing phosphorylation sites in sirtuin 3 (pS103) and sirtuin 1 (pS670) with 14-3-3 proteins. We further describe the crystal structures of complexes of 14-3-3σ with either of the two phosphopeptides. As a conclusion, we propose a more extended 14-3-3 binding mode on the N-terminal side of the phosphorylation site and the possibility of nongeneric motifs and conformations on the C-terminal side, still resulting in the known high binding affinity of the two partners.
14-3-3 PROTEINS; SIRTUINS; PHOSPHOPEPTIDES; REGULATION; PROTEIN-PROTEIN INTERACTION
Crystal structure of Methanococcus jannaschii dihydroorotase with substrate bound
Here, we report the X-ray structural analysis of dihydroorotase from Methanococcus jannaschii co-crystallized with dihydroorotate at pH 6.5. The crystals are isomorphous with the crystals of the apoenzyme, with space group P3221 and unit-cell dimensions a = b = 111.4, c = 101.2 Å. The structure was refined to R = 0.169 and Rfree = 0.186 at a resolution of 1.87 Å at room temperature. During crystallization the degradative reaction took place, and the electron density in the active site corresponds to the substrate carbamoyl aspartate. Carbamoyl aspartate interacts with the protein in the active site in a manner similar to that observed for Escherichia coli and human dihydroorotases. However, the flexible loop (residues 140–151) adopts both conformations in the crystal, loop-out and loop-in, with the loop-out conformation having higher occupancy. This contrasts with our expectations for the flexible loop to be exclusively in the loop-in conformation, which is the conformation that it adopts to stabilize the binding of the substrate in the known systems. Additional studies with substrate analogs that resemble carbamoyl aspartate and different crystallization conditions would provide further insight into the conformation of the flexible loop in this system.
DIHYDROOROTASE; METHANOCOCCUS JANNASCHII; PYRIMIDINE BIOSYNTHESIS; FLEXIBLE LOOP
Single mutations to tyrosine or glutamate improve the crystallizability and crystal diffraction properties of a flexible two-domain protein
This case report describes single surface substitutions that improve the crystallizability and diffraction properties of a flexible two-domain protein. InlB392 comprises the internalin domain and the B repeat of the Listeria monocytogenes invasion protein InlB. The InlB392 wild type yielded very few poorly reproducible hits in crystallization screens and the crystals had a diffraction limit of worse than 3.0 Å. It seems reasonable to assume that this crystallization bottleneck is caused by interdomain flexibility, given that crystals of the isolated internalin domain or B repeat diffract to high resolution. A previously identified variant, T332E, showed improved crystallization and diffraction. Here, two additional InlB392 variants are described with single threonine-to-tyrosine or valine-to-glutamate substitutions that produced crystals directly in initial screens and, without optimization, diffracted to 1.6 and 1.45 Å resolution, respectively. The mutated residues do not participate in intramolecular interdomain interactions but mediate crystal contacts, indicating that specific surface properties, rather than interdomain flexibility per se, impede the crystallization of wild-type InlB392. Notably, the beneficial glutamate substitutions contrast with the generally recognized underrepresentation of glutamate in crystal contacts and the high entropic cost of fixing an otherwise flexible side chain with many rotatable bonds in a crystal contact. The reported results suggest that surface mutations can help crystallization even if they increase the entropy of the respective residue. More broadly, the observations are consistent with the hypothesis that negative evolutionary design limits fortuitous lattice formation of proteins and the resulting expectation that random mutations of surface residues are likely to improve crystallizability.
MULTIDOMAIN PROTEINS; POINT MUTATIONS; PROTEIN CRYSTALLIZATION; SURFACE-ENTROPY REDUCTION; SURFACE MUTATION; CRYSTAL CONTACTS
A note on the appearance of PEG in macromolecular crystals
POLYETHYLENE GLYCOL; MECHANISM; CRYSTALLIZATION; REFINEMENT; MODELING
Crystal structure of a short-chain dehydrogenase from Brucella ovis with apo and coenzyme NAD+-bound protomer chains
Short-chain dehydrogenases (SDRs) are a family of NAD(P)-dependent enzymes involved in redox reactions, specifically carbonyl–alcohol reductions. Here, we report the apo and NAD+-bound structures of an SDR from the pathogenic organism Brucella ovis. B. ovis primarily affects sheep and other livestock, resulting in reduced fertility. Based on sequence and structural alignment, the B. ovis SDR (BoSDR) is a classical SDR. Classical SDRs have a canonical YxxxK active-site sequence in which the catalytic general base is a tyrosine residue located at position 163. In addition, the putative active site also contains a serine residue (Ser150) and lysine residue (Lys167) that are hypothesized to be involved in catalysis. BoSDR is a biological and crystallographic tetramer. In the coenzyme-bound structure, two different orientations of the NAD+ coenzyme are fortuitously observed, which provides insights into the conformational changes that accompany coenzyme binding. The apo and NAD+-bound structures provide valuable information about the unique structural features of enzymes in the SDR superfamily.
SHORT-CHAIN DEHYDROGENASE/REDUCTASES; RESEARCH EXPERIENCE FOR UNDERGRADUATES; REDOX CHEMISTRY; COENZYME BINDING; SSGCID
High-resolution X-ray structure of Gln143Asn manganese superoxide dismutase captures multiple hydrogen peroxide-binding sites
Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide into hydrogen peroxide and molecular oxygen, serving as a key defence against oxidative damage. Despite extensive studies, the full structural characterization of H2O2-binding sites in MnSOD remains largely unexplored. Previous H2O2-soaked MnSOD structures have identified two distinct H2O2-binding sites: one directly ligated to the catalytic manganese (LIG position) and another at the active-site gateway (PEO position) between the second-shell residues Tyr34 and His30. In this study, a kinetically impaired Gln143Asn MnSOD variant is used to trap and explore additional H2O2-binding sites beyond the second-shell solvent gate. In the wild-type enzyme, Gln143 mediates proton transfers with the manganese-bound solvent (WAT1) to drive redox cycling of the metal, which is necessary for effective superoxide dismutation. Substitution with Asn stalls catalysis because the increased distance from WAT1 disrupts critical proton-coupled electron-transfer (PCET) events, and the redox cycling of the active-site metal is impaired. This, in turn, stalls the electrostatic cycling of positive charge on the enzyme surface and enhances the likelihood of trapping transient H2O2-bound states in this variant. The results reveal several H2O2 molecules leading up to the active site, in addition to the canonical LIG and PEO positions.
MANGANESE SUPEROXIDE DISMUTASES; PEROXIDE BINDING; METALLOENZYMES; X-RAY CRYSTALLOGRAPHY
CryoSift: an accessible and automated CNN-driven tool for cryo-EM 2D class selection
Single-particle cryo-electron microscopy (cryo-EM) has become an essential tool in structural biology. However, automating repetitive tasks remains an ongoing challenge in cryo-EM data-set processing. Here, we present a platform-independent convolutional neural network (CNN) tool for assessing the quality of 2D averages to enable the automatic selection of suitable particles for high-resolution reconstructions, termed CryoSift. We integrate CryoSift into a fully automated processing pipeline using the existing cryosparc-tools library. Our integrated and customizable 2D assessment workflow enables high-throughput processing that accommodates experienced to novice cryo-EM users.
AUTOMATION; DATA PROCESSING; CRYO-EM; SINGLE-PARTICLE CRYO-EM; DEEP LEARNING
Automated gradient equilibration of macromolecular crystals to new solution conditions
We describe a device and a method for changing the ambient solution of a macromolecular crystal. The approach is gentle, automated, inexpensive and open source. Examples are given of the equilibration of three different crystals to new solutions with exchange times ranging from 5 to 180 min. In each case direct transfer of the crystal to the new solution causes cracking, which is eliminated with gradient equilibration using the described device. Crystals equilibrated with the device produce high-quality diffraction that yields refined structures comparable to those determined previously. The device offers a more systematic and labor-saving workflow than current practice both for performing diffraction analysis of macromolecular crystals and for investigating the response of macromolecular crystals to changes in solution composition.
PERC: a suite of software tools for the curation of cryoEM data with application to simulation, modeling and machine learning
Ease of access to data, tools and models expedites scientific research. In structural biology there are now numerous open repositories of experimental and simulated data sets. Being able to easily access and utilize these is crucial to allow researchers to make optimal use of their research effort. The tools presented here are useful for collating existing public cryoEM data sets and/or creating new synthetic cryoEM data sets to aid the development of novel data processing and interpretation algorithms. In recent years, structural biology has seen the development of a multitude of machine-learning-based algorithms to aid numerous steps in the processing and reconstruction of experimental data sets and the use of these approaches has become widespread. Developing such techniques in structural biology requires access to large data sets, which can be cumbersome to curate and unwieldy to make use of. In this paper, we present a suite of Python software packages, which we collectively refer to as PERC (profet, EMPIARreader and CAKED). These are designed to reduce the burden which data curation places upon structural biology research. The protein structure fetcher (profet) package allows users to conveniently download and cleave sequences or structures from the Protein Data Bank or AlphaFold databases. EMPIARreader allows lazy loading of Electron Microscopy Public Image Archive data sets in a machine-learning-compatible structure. The Class Aggregator for Key Electron-microscopy Data (CAKED) package is designed to seamlessly facilitate the training of machine-learning models on electron microscopy data, including electron-cryo-microscopy-specific data augmentation and labeling. These packages may be utilized independently or as building blocks in workflows. All are available in open-source repositories and designed to be easily extensible to facilitate more advanced workflows if required.
CRYOEM; ELECTRON CRYOMICROSCOPY; PYTHON; PERC
Off-target binding of the histone deacetylase inhibitor vorinostat to carbonic anhydrase II and IX
Histone deacetylase inhibitors (HDACi) are widely used in cancer therapy but often suffer from off-target effects due to their pan-inhibitory activity towards zinc-dependent enzymes. Vorinostat (SAHA), a hydroxamate-based HDACi, has been shown to lack isoform selectivity, potentially leading to unintended interactions with other metalloenzymes. Here, we report high-resolution crystal structures of SAHA bound to human carbonic anhydrase II (CA II) and a carbonic anhydrase IX (CA IX) active-site mimic. Structures determined at room temperature and 100 K revealed two distinct SAHA conformers in both CA II and the CA IX mimic, with the hydroxamate moiety displacing the zinc-bound water and adopting either a tetrahedral or pentahedral coordination to Zn2+. Differences in hydrophobic interactions were observed between CA II and the CA IX mimic due to the F131V amino-acid difference between the two enzymes. SwissDock modeling accurately predicted the SAHA binding orientations observed in crystallography. Thermal shift assays using nanoDSF showed minimal stabilization of either CA by SAHA, in contrast to the potent CA inhibitor acetazolamide. Binding-energy calculations suggest that SAHA may bind carbonic anhydrases with affinities comparable to its HDAC targets. These findings highlight potential off-target binding of SAHA to carbonic anhydrases, which may contribute to its clinical side effects. The results also suggest that hydroxamates may serve as a nonsulfonamide scaffold for novel CA inhibitors, although isoform selectivity remains a challenge.
HDAC INHIBITORS; SAHA; VORINOSTAT; HUMAN CARBONIC ANHYDRASES; OFF-TARGET BINDING
Crystal structure of a seven-substitution mutant of hydroxynitrile lyase from rubber tree
The α/β-hydrolase fold superfamily includes esterases and hydroxynitrile lyases which, despite catalyzing different reactions, share a Ser–His–Asp catalytic triad. We report a 1.99 Å resolution crystal structure of HNL6V, an engineered variant of hydroxynitrile lyase from Hevea brasiliensis (HbHNL) containing seven amino-acid substitutions (T11G, E79H, C81L, H103V, N104A, G176S and K236M). The structure reveals that HNL6V maintains the characteristic α/β-hydrolase fold while exhibiting systematic shifts in backbone and catalytic atom positions. Compared with wild-type HbHNL, the Cα positions in HNL6V differ by a mean of 0.2 ± 0.1 Å, representing a statistically significant displacement. Importantly, the catalytic triad and oxyanion-hole atoms have moved 0.2–0.8 Å closer to their corresponding positions in SABP2, although they remain 0.3–1.1 Å from fully achieving the configuration of SABP2. The substitutions also increase local flexibility, particularly in the lid domain covering the active site. This structural characterization demonstrates that targeted amino-acid substitutions can systematically shift catalytic geometries towards those of evolutionarily related enzymes.
ENGINEERED PROTEINS; HYDROXYNITRILE LYASES; [ALPHA]/[BETA]-HYDROLASE FOLD; ESTERASES
Crystal structure of the folded domains of Xrs2 from Saccharomyces cerevisiae
The MRE11–RAD50–NBS1/Xrs2 (MRN/X) protein complex acts as a first responder in DNA double-strand break repair and telomere-length maintenance, yet the structural architecture of the yeast ortholog Xrs2 has remained unresolved. In this study, we present the first structure of the folded N-terminal region of Xrs2 from Saccharomyces cerevisiae, resolved at 2.38 Å using X-ray crystallography. Like the previously determined crystal structures of Schizosaccharomyces pombe Nbs1, the folded structure of S. cerevisiae Xrs2 adopts an extended three-domain organization at its N-terminus. Electrostatic analysis reveals two distinct charged patches: a positively charged patch on the FHA domain and a negatively charged patch in the cleft between the FHA and BRCT1 domains. This charge segregation is likely to play a role in mediating interactions with various ligands.
NBS1/XRS2; DNA DOUBLE-STRAND BREAKS; DNA DAMAGE REPAIR
Triosephosphate isomerase from Fasciola hepatica: high-resolution crystal structure as a drug target
The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.
FASCIOLA HEPATICA; TRIOSEPHOSPHATE ISOMERASE; TPI; TIM; TRICLABENDAZOLE; CRYSTAL STRUCTURE
Structures of Listeria monocytogenes MenD in ThDP-bound and in-crystallo captured intermediate I-bound forms
Menaquinones (vitamin K2) are a family of redox-active small lipophilic molecules that serve as vital electron carriers in many bacterial electron-transport pathways. The ThDP-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase (MenD) catalyses the first irreversible step in bacterial classical menaquinone biosynthesis via a series of reactions involving covalent ThDP-bound intermediates. We report structures of MenD from the pathogen Listeria monocytogenes (LmoMenD) in its ThDP cofactor-bound and in-crystallo captured intermediate I-bound forms. Analysis of the structures revealed that LmoMenD adopts the typical three-domain ThDP-dependent fold observed for MenD orthologs, while a combination of structure, size-exclusion chromatography, mass photometry and small-angle X-ray scattering analysis showed that the enzyme has a homotetrameric quaternary structure. While both of the ligand-bound structures reported here were very similar, comparison with an apo form from the PDB revealed a closing down of the active site in the ligand-bound forms, with more complete models suggesting lower levels of disorder around key regions of the active site that interface with ThDP or the captured intermediate. Enzyme kinetics characterization showed the enzyme was active and enabled allosteric inhibition to be measured. There was weak inhibition of enzyme activity in the presence of 1,4-dihydroxy-2-naphthoic acid, an allosteric regulator of Mycobacterium tuber­culosis MenD and downstream metabolite in the menaquinone-biosynthesis pathway.
LISTERIA MONOCYTOGENES; MENAQUINONE BIOSYNTHESIS; SEPHCHC SYNTHASE; MEND; THIAMINE DIPHOSPHATE-DEPENDENT ENZYME; INTERMEDIATE I CAPTURE
Crystal structures of Escherichia coli glucokinase and insights into phosphate binding
Here, we report the crystal structure of Escherichia coli glucokinase (GLK), which has phosphate bound in the cleft between the α and β domains adjacent to the active site. A ternary complex consisting of GLK, glucose and phosphate is also reported in this work. Diffraction data were collected at 2.63 Å resolution for the phospate-bound form (Rwork/Rfree = 0.191/0.230) and at 2.54 Å resolution for the ternary complex (Rwork/Rfree = 0.202/0.258), both at 297 K. A B-factor analysis of the phosphate-bound GLK structure revealed consistently lower values for phosphate-interacting basic residues in the α4, α5 and α9 helices, while significant root-mean-square deviation (r.m.s.d.) spikes indicated flexibility in regions preceding β1 and within the loop between the β5 and β6 sheets of the α domain. In the ternary complex, phosphate is bound adjacent to glucose, and the B factors for the α4, α5 and α9 helices were further reduced, while r.m.s.d. spikes were observed at the end of the β10 sheet and within the α6 helix of the β-domain. This structural characterization suggests that phosphate could influence the activity of GLK by altering glucose binding and modulating interactions with a loop-interacting regulatory protein.
GLUCOKINASES; HEXOKINASES; PHOSPHATE BINDING; SULFATE BINDING
Nucleotide-bound crystal structures of the SARS-CoV-2 helicase NSP13
Nucleotide-bound crystal structures of SARS-CoV-2 NSP13 in ADP- and ATP-bound states were resolved to 1.8 and 1.9 Å, respectively. The ADP-bound model captures a state immediately following ATP hydrolysis, with both ADP and orthophosphate still present in the active site. Further comparative analysis revealed that crystal packing influences NSP13 by stabilizing the nucleotide-binding site, underscoring the importance of accounting for these effects in structure-based drug design targeting NSP13.
SARS-COV-2; NSP13 HELICASE; NUCLEOTIDE-BINDING SITES; INORGANIC PHOSPHATE; ADP-BOUND STRUCTURE; ATP-BOUND STRUCTURE; COVID-19
Structural insights into full-length human fascin1: a target for cancer treatment
Fascin1 proteins are a family of globular proteins with actin-bundling activity that cross-link actin filaments together, allowing the formation of actin-rich structures involved in cell migration and adhesion, such as filopodia, invadopodia, stress fibers, micro-spikes and podocytes. The overexpression of human fascin1 has been linked to tumor progression in most human cancers, particularly during the epithelial–mesenchymal transition, making it a promising biomarker for cancer metastasis and a major target for the development of novel cancer therapies. X-ray crystallography has been instrumental in human fascin1-inhibition research since it provides detailed insights into the structure of the protein and its interactions with small-molecule inhibitors. This technique has allowed the characterization of a range of molecular conformations in which the protein naturally exists. However, human fascin1 has never been fully modeled until now. To the best of our knowledge, this study presents the first full-length structure of human fascin1 in which both copies are fully resolved. Comparison of this structure with the available wild-type and complexed structures provides new insights into the conformational plasticity of fascin1 that will facilitate subsequent studies on human fascin1 in the context of drug design for cancer-related therapies.
HUMAN FASCIN1; CANCER; METASTASIS; DYNAMICS; PLASTICITY; ACTIN-BINDING PROTEINS; X-RAY CRYSTALLOGRAPHY
Functional and structural characterization of Stenotrophomonas maltophilia EntB, an unusual form of isochorismatase for siderophore synthesis
Clinical and environmental isolates of Stenotrophomonas maltophilia produce an enterobactin-like siderophore that promotes bacterial growth under low-iron conditions. Although prior mutational and bioinformatic analyses indicated that most of the enzymes encoded by the S. maltophilia entCEBB′FA locus are suitably reminiscent of their counterparts in Escherichia coli and other bacteria, Stenotrophomonas EntB was unusual. In bacteria producing enterobactin-related molecules, EntB and its homologs are usually multi-domain proteins in which the amino portion acts as an isochorismatase and the carboxy domain serves as an aryl carrier protein (ArCP). However, in S. maltophilia the isochorismatase and ArCP functions are encoded by two distinct genes: entB and entB′, respectively. Current mutant analysis was used to first confirm that S. maltophilia entB is needed for siderophore activity and bacterial growth in iron-depleted media. A crystal structure of S. maltophilia EntB was then obtained. The structure aligned with the N-terminal portion of EntB from E. coli and VibB from Vibrio cholerae, affirming the protein to be a single-domain isochorismatase. However, S. maltophilia EntB also aligned with the single-domain PhzD from Pseudomonas aeruginosa, which is a key enzyme involved in the biosynthesis of the antimicrobial compound phenazine. BLASTP searches indicated that entB and its neighboring genes are fully conserved amongst S. maltophilia strains but are variably present in other Stenotrophomonas species. The closest homologs to S. maltophilia EntB outside the genus were hypothetical proteins/putative isochorismatases in some Gram-negative bacteria (for example Pseudomonas spp. and Xanthomonas spp.), Gram-positive bacteria (Streptomyces spp. and Bacillus subtilis) and fungi (for example Rhizopus arrhizus and Knufia peltigerae).
STENOTROPHOMONAS MALTOPHILIA; SIDEROPHORES; ENTEROBACTIN; ISOCHORISMATASES; ENTB; ISOCHORISMATE LYASES
Preparing for successful protein crystallization experiments
Crystal-based structural methods, including X-ray crystallography, are frequently utilized for the determination of high-resolution structures of biomolecules. All crystal-based diffraction methods first require the preparation of biomolecular crystals, and careful sample preparation for crystallization experiments can increase the frequency of success. In this article, strategies to optimize factors that can impact crystallization are presented, from which buffers and reducing agents are most favorable to which crystallization techniques could be used.
CRYSTALLIZATION; X-RAY CRYSTALLOGRAPHY; STRUCTURAL BIOLOGY; X-RAY FREE-ELECTRON LASERS; ELECTRON DIFFRACTION
Crystal structure of the sucrose phosphorylase from Alteromonas mediterranea shows a loop transition in the active site
Sucrose phosphorylases are essential enzymes regulating sucrose metabolism, and it has been shown that a loop rearrangement is essential to their catalytic cycle. Crystal structures of only six sucrose phosphorylase enzymes are available. Here, we present the crystal structure of a sucrose phosphorylase from a proteobacterium, Alteromonas mediterranea, at 2.15 Å resolution. The available sucrose phosphorylase structures have shown that an important conformational change occurs during the catalytic cycle or upon mutagenesis. Interestingly, our data present clear indications of the two major conformations in the same crystal.
SUCROSE PHOSPHORYLASES; GH13; MARINE; LOOP
The structure of a family 168 glycoside hydrolase from the marine bacterium Muricauda eckloniae
The genome of the marine bacterium Muricauda eckloniae sp. DK169 contains an extensive polysaccharide-utilization locus that targets fucoidan from brown algae. Within this locus is a gene that encodes a putative fucoidan-degrading glycoside hydrolase (locus tag AAY42_01205) assigned to glycoside hydrolase family 168, which we call MeGH168. We present the 2.0 Å resolution X-ray crystal structure of MeGH168, demonstrating a (β/α)8-barrel fold. The eight loop regions joining each α-helix and β-strand surround the catalytic groove. A comparison with the structure of a GH168, Fun168A, in complex with a fragment of fucoidan (PDB entry 8ya7) revealed conservation of key residues in the catalytic site. However, structural variation in positive-subsite loop regions may recontour the active site to create differences in substrate specificity between the two GH168s. The present data provide additional structural insights into the GH168 family, particularly expanding on sequence and structure conservation (and the lack thereof) in relation to substrate interactions.
GLYCOSIDE HYDROLASES; FUCOIDANASES; MARINE BACTERIA; MURICAUDA ECKLONIAE
Crystal structure of ATP-dependent DNA ligase from Rhizobium phage vB_RleM_P10VF
DNA ligases are foundational molecular-biological tools used for cloning and sequencing workflows, and are essential replicative enzymes for all cellular life forms as well as many viruses and bacteriophage. There is considerable interest in structurally and functionally characterizing novel DNA ligases and profiling their suitability for molecular-biological applications. Here, we report the crystal structure of the ATP-dependent DNA ligase from the Rhizobium phage vB_RleM_P10VF bound to a nicked DNA duplex determined to 2.2 Å resolution. The enzyme crystallized in the DNA-encircling conformation, arrested as a step 2 intermediate in the catalytic cycle with the adenylating cofactor transferred to the 5′-phosphate of the DNA nick. The overall structure of the DNA ligase closely resembles that of the T4 DNA ligase, including an α-helical globular DNA-binding domain. Several secondary-structural elements are abbreviated in the P10VF DNA ligase relative to the T4 DNA ligase enzyme, which may account for its lower specific activity, especially on DNA substrates containing double-stranded breaks.
Structural analysis of YcdY, a member of the redox-enzyme maturation protein family
Proteins of the NarJ subfamily from facultatively or obligately anaerobic bacteria play key roles as chaperones in folding and cofactor insertion for complex iron–sulfur molybdoenzymes (CISMs), which mediate energy production under anaerobic conditions. YcdY was identified as a NarJ subfamily member but was proposed to increase the catalytic activity of the non-CISM enzyme YcdX phosphatase, presumably by inserting a zinc cofactor into YcdX. To elucidate the structural features of YcdY required for its chaperone function, we determined the crystal structure of Enterobacter cloacae YcdY (enYcdY). enYcdY adopts a single-domain, curved helix-bundle structure decorated with α-helices. enYcdY contains an extensive dent on its concave side. The dent in enYcdY generally forms using hydrophobic or conserved residues. Based on comparative structural and sequence analyses, we propose that enYcdY uses the dent to recognize and fold the client protein. Interestingly, enYcdY did not increase the enzymatic activity of E. cloacae YcdX (enYcdX) in the presence or absence of Zn2+ ions, even for partially denatured enYCdX protein. The same results were obtained for the Escherichia coli counterparts, in contrast to a previous report. These observations suggest that YcdY functions as a chaperone for proteins other than YcdX.
YCDY; YCDX; ENTEROBACTER CLOACAE; CHAPERONES
Structure of the Saccharolobus solfataricus GINS tetramer
DNA replication is tightly regulated to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance (MCM) helicase rings that unwind DNA during its replication. In eukaryotes, MCM activation critically relies on the sequential recruitment of the essential factors Cdc45 and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex. We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus (Sso) to reveal a core structure that is highly similar to the previously determined GINS core structures of other eukaryotes and archaea. Using molecular modeling, we illustrate that a subdomain of SsoGINS would need to move to accommodate known interactions of the archaeal GINS complex and to generate a SsoCMG complex analogous to that of eukaryotes.
DNA REPLICATION; GINS; MINICHROMOSOME MAINTENANCE; ARCHAEA
Structures of Mycobacterium tuberculosis isoprenyl diphosphate synthase Rv2173 in substrate-bound forms
We report structures of the Mycobacterium tuberculosis isoprenyl diphosphate synthase Rv2173 in three forms: apo and two substrate-bound forms [isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)]. The protein possesses a canonical all-α-helical trans-isoprenyl diphosphate synthase fold that is dimeric in each form. There are some differences between the structures: the IPP-bound form shows IPP bound in the DMAPP/allylic substrate-binding site with three divalent metal ions bound around the IPP and the complete C-terminus closing around the active site, while the apo and DMAPP-bound forms are more open, with some of the C-terminal region disordered, supporting suggestions that the C-terminus is important in substrate entry/product exit. In the DMAPP form DMAPP occupies the expected allylic substrate site, but only two metal ions are associated with the binding, with the DMAPP diphosphates adopting a slightly different binding pose compared with IPP in the same site, and the third metal-binding site is unoccupied. In no case is the IPP binding site occupied by IPP. There has been some uncertainty regarding product length for Rv2173, with variable lengths being reported. In the structures reported here, the `capping' residue at the bottom of the binding cavity is tryptophan and comparison with other IPP synthases suggests that the structure of Rv2173 is most consistent with a C10–C15 final product size.
MYCOBACTERIUM TUBERCULOSIS; ISOPRENYL DIPHOSPHATE SYNTHASES; RV2173; DIMETHYLALLYL DIPHOSPHATE; ISOPRENYL DIPHOSPHATE
The structure of the Gemella haemolysans M26 IgA1 protease trypsin-like domain
Immunoglobulin A (IgA) proteases are a group of bacterial-derived enzymes that selectivity hydrolyze human IgA in the hinge region that is unique to this immunoglobulin. Several IgA protease (IgAP) families have evolved this ability using both metalloprotease and serine protease chemical mechanisms. One family of metal-dependent IgAPs is the M26 family. This family can be grouped into two subfamilies based upon the presence or absence of a trypsin-like domain found N-terminal to the IgAP domain. The role of this domain in IgAP structure and function is poorly understood. Here, we present the first structural characterization of an M26 IgAP trypsin-like domain from Gemella haemo­lysans (GhTrp). These structural data demonstrate that the GhTrp domain possesses a trypsin-like fold but contains significant deviations in the surface-loop structure that is known to be coupled to protease selectivity. The lack of observable catalytic function coupled with the structural data suggest that this domain may exist in a pro-enzyme-like state that can potentially be activated when the domain is N-terminally proteolytically excised from the larger M26 IgAP structure.
IGA PROTEASES; SERINE PROTEASES; TRYPSIN; CHYMOTRYPSIN; M26 PROTEASES
Cryo-EM structures of apo and atorvastatin-bound human 3-hydroxy-3-methylglutaryl-coenzyme A reductase
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) regulates the level of cholesterol by catalysing the formation/production of mevalonate and has therefore become an important pharmaceutical target for coronary heart disease. Here, we report the cryo-EM structure of the catalytic part of the enzyme in the apo form and bound with its inhibitor atorvastatin, a commonly used drug in cardiovascular disease, at resolutions of 2.1 and 2.3 Å, respectively. In the cryo-EM maps, part of the N-domain corresponding to amino acids 439–487 is well ordered and could be modelled completely. Atorvastatin molecules were found to occupy all four active sites of the tetrameric complex, and the binding does not alter the conformation of the protein or the active site. The method described here exploits graphene oxide as an additional support and could be used as an alternative to elucidate the structures of pharmaceutical target compounds that are difficult to co-crystallize with human HMGR and for sparsely available samples in drug discovery.
SPA CRYO-EM; HMGR; ATORVASTATIN; GRAPHENE OXIDE
LOPAC library screening identifies suramin as a TRIM21 binder with a unique binding mode revealed by crystal structure
Differential scanning fluorimetry screening of the Library of Pharmacologically Active Compounds (LOPAC) identified four hits for the PRYSPRY domain of the human E3 ligase tripartite motif-containing protein 21 (TRIM21). Isothermal titration calorimetry subsequently confirmed suramin as a binder with micromolar affinity. To further investigate the binding mechanism, mouse TRIM21 was used as a structural surrogate due to its improved protein stability and high sequence similarity to the human counterpart. A crystal structure of the complex refined at 1.3 Å resolution revealed a unique binding mode, providing new avenues for targeting TRIM21 and for the development of proteolysis-targeting chimeras (PROTACs).
TRIM21; E3 LIGASES; CRYSTAL STRUCTURE; SURAMIN