Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin.

Rhodanese domains are structural modules present in the sulfurtransferase superfamily. These domains can exist as single units, in tandem repeats, or fused to domains with other activities. Despite their prevalence across species, the specific physiological roles of most sulfurtransferases are not known. Mammalian rhodanese and mercaptopyruvate sulfurtransferase are perhaps the best-studied members of this protein superfamily and are involved in hydrogen sulfide metabolism. The relatively unstudied human thiosulfate sulfurtransferase-like domain-containing 1 (TSTD1) protein, a single-domain cytoplasmic sulfurtransferase, was also postulated to play a role in the sulfide oxidation pathway using thiosulfate to form glutathione persulfide, for subsequent processing in the mitochondrial matrix. Prior kinetic analysis of TSTD1 was performed at pH 9.2, raising questions about relevance and the proposed model for TSTD1 function. In this study, we report a 1.04 Å resolution crystal structure of human TSTD1, which displays an exposed active site that is distinct from that of rhodanese and mercaptopyruvate sulfurtransferase. Kinetic studies with a combination of sulfur donors and acceptors reveal that TSTD1 exhibits a low Km for thioredoxin as a sulfane sulfur acceptor and that it utilizes thiosulfate inefficiently as a sulfur donor. The active site exposure and its interaction with thioredoxin suggest that TSTD1 might play a role in sulfide-based signaling. The apical localization of TSTD1 in human colonic crypts, which interfaces with sulfide-releasing microbes, and the overexpression of TSTD1 in colon cancer provide potentially intriguing clues as to its role in sulfide metabolism.

Structure-guided insights on the role of NS1 in flavivirus infection.

We highlight the various domains of the flavivirus virulence factor NS1 and speculate on potential implications of the NS1 3D structure in understanding its role in flavivirus pathogenesis. Flavivirus non-structural protein 1 (NS1) is a virulence factor with dual functions in genome replication and immune evasion. Crystal structures of NS1, combined with reconstructions from electron microscopy (EM), provide insight into the architecture of dimeric NS1 on cell membranes and the assembly of a secreted hexameric NS1-lipid complex found in patient sera. Three structural domains of NS1 likely have distinct roles in membrane association, replication complex assembly, and immune system avoidance. A conserved hydrophobic inner face is sequestered either on the membrane or in the interior of the secreted hexamer and contains regions implicated in viral replication. The exposed variable outer face is presented to cellular and secreted components of the immune system in infected patients and contains candidate regions for immune system modulation. We anticipate that knowledge of the distinct NS1 domains and assembly will lead to advances in elucidating virus-host interactions mediated through NS1 and in dissecting the role of NS1 in viral genome replication.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly.

UNLABELLED: The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1' position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1' position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1' position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. IMPORTANCE: West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific antiflavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this article, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies.

The oligomeric state of CtBP determines its role as a transcriptional co-activator and co-repressor of Wingless targets.

C-terminal-binding protein (CtBP) is a well-characterized transcriptional co-repressor that requires homo-dimerization for its activity. CtBP can both repress and activate Wingless nuclear targets in Drosophila. Here, we examine the role of CtBP dimerization in these opposing processes. CtBP mutants that cannot dimerize are able to promote Wingless signalling, but are defective in repressing Wingless targets. To further test the role of dimerization in repression, the positions of basic and acidic residues that form inter-molecular salt bridges in the CtBP dimerization interface were swapped. These mutants cannot homo-dimerize and are compromised for repression. However, their co-expression leads to hetero-dimerization and consequent repression of Wingless targets. Our results support a model where CtBP is a gene-specific regulator of Wingless signalling, with some targets requiring CtBP dimers for inhibition while other targets utilize CtBP monomers for activation of their expression. Functional interactions between CtBP and Pygopus, a nuclear protein required for Wingless signalling, support a model where monomeric CtBP acts downstream of Pygopus in activating some Wingless targets.

Use of massively multiple merged data for low-resolution S-SAD phasing and refinement of flavivirus NS1.

An emergent challenge in macromolecular crystallography is the identification of the substructure from native anomalous scatterers in crystals that diffract to low to moderate resolution. Increasing the multiplicity of data sets has been shown to make previously intractable phasing problems solvable and to increase the useful resolution in model refinement. For the West Nile virus nonstructural protein 1 (NS1), a protein of novel fold, the utility of exceptionally high multiplicity data is demonstrated both in solving the crystal structure from the anomalous scattering of the native S atoms and in extending the useful limits of resolution during refinement. A high-multiplicity data set from 18 crystals had sufficient anomalous signal to identify sulfur sites using data to 5.2 Šresolution. Phases calculated to 4.5 Šresolution and extended to 3.0 Šresolution were of sufficient quality for automated building of three-quarters of the final structure. Crystallographic refinement to 2.9 Šresolution proceeded smoothly, justifying the increase in resolution that was made possible by combining multiple data sets. The identification and exclusion of data from outlier crystals is shown to result in more robust substructure determination.

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.

Structure of a modular polyketide synthase reducing region.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor.

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

Insights from the sea: structural biology of marine polyketide synthases.

The world's oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe(2+)/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for ß-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

A general scheme for synthesis of substrate-based polyketide labels for acyl carrier proteins.

A general strategy to enzymatically label acyl carrier proteins (ACPs) of polyketide synthases has been developed. Incorporation of a chloromethyl ketone or vinyl ketone moiety into polyketide chain elongation intermediate mimics allows for the synthesis of CoA adducts. These CoA adducts undergo enzymatic reaction with Sfp, a phosphopantetheinyl transferase, to afford labeled CurB carrier proteins.

Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis.

Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.

Cryo-EM Analysis Reveals Structural Basis of Helicobacter pylori VacA Toxin Oligomerization.

Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single-particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8-Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.

Efficient merging of data from multiple samples for determination of anomalous substructure.

Merging of data from multiple crystals has proven to be useful for determination of the anomalously scattering atomic substructure for crystals with weak anomalous scatterers (e.g. S and P) and/or poor diffraction. Strategies for merging data from many samples, which require assessment of sample isomorphism, rely on metrics of variability in unit-cell parameters, anomalous signal correlation and overall data similarity. Local scaling, anomalous signal optimization and data-set weighting, implemented in phenix.scale_and_merge, provide an efficient protocol for merging data from many samples. The protein NS1 was used in a series of trials with data collected from 28 samples for phasing by single-wavelength anomalous diffraction of the native S atoms. The local-scaling, anomalous-optimization protocol produced merged data sets with higher anomalous signal quality indicators than did standard global-scaling protocols. The local-scaled data were also more successful in substructure determination. Merged data quality was assessed for data sets where the multiplicity was reduced in either of two ways: by excluding data from individual crystals (to reduce errors owing to non-isomorphism) or by excluding the last-recorded segments of data from each crystal (to minimize the effects of radiation damage). The anomalous signal was equivalent at equivalent multiplicity for the two procedures, and structure-determination success correlated with anomalous signal metrics. The quality of the anomalous signal was strongly correlated with data multiplicity over a range of 12-fold to 150-fold multiplicity. For the NS1 data, the local-scaling and anomalous-optimization protocol handled sample non-isomorphism and radiation-induced decay equally well.

Extended surface for membrane association in Zika virus NS1 structure.

The Zika virus, which has been implicated in an increase in neonatal microcephaly and Guillain-Barré syndrome, has spread rapidly through tropical regions of the world. The virulence protein NS1 functions in genome replication and host immune-system modulation. Here, we report the crystal structure of full-length Zika virus NS1, revealing an elongated hydrophobic surface for membrane association and a polar surface that varies substantially among flaviviruses.

Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing.

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.

Can I solve my structure by SAD phasing? Planning an experiment, scaling data and evaluating the useful anomalous correlation and anomalous signal.

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilliger et al. (2016), Acta Cryst. D72, 346-358] is used to develop methods for planning a SAD experiment, scaling SAD data sets and estimating the useful anomalous correlation and anomalous signal in a SAD data set. The phenix.plan_sad_experiment tool uses a database of solved and unsolved SAD data sets and the expected characteristics of a SAD data set to estimate the probability that the anomalous substructure will be found in the SAD experiment and the expected map quality that would be obtained if the substructure were found. The phenix.scale_and_merge tool scales unmerged SAD data from one or more crystals using local scaling and optimizes the anomalous signal by identifying the systematic differences among data sets, and the phenix.anomalous_signal tool estimates the useful anomalous correlation and anomalous signal after collecting SAD data and estimates the probability that the data set can be solved and the likely figure of merit of phasing.

Structure of the nuclease domain of ribonuclease III from M. tuberculosis at 2.1 A.

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded (ds)RNA. These proteins are involved in a diverse group of functions, including ribosomal RNA processing, mRNA maturation and decay, snRNA and snoRNA processing, and RNA interference. Here we report the crystal structure of the nuclease domain of RNase III from the pathogen Mycobacterium tuberculosis. Although globally similar to other RNase III folds, this structure has some features not observed in previously reported models. These include the presence of an additional metal ion near the catalytic site, as well as conserved secondary structural elements that are proposed to have functional roles in the recognition of dsRNAs.

Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.