Jane A. Potter

The Structure of Clostridium perfringens NanI Sialidase and Its Catalytic Intermediates

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97Å resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97Å resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5Å resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2Å resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain.

BackgroundIPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region.ResultsThe crystal structure of human IPS-1/MAVS/VISA/Cardif CARD has been determined to 2.1Å resolution. The protein was expressed and crystallized as a maltose-binding protein (MBP) fusion protein. The MBP and IPS-1 components each form a distinct domain within the structure. IPS-1/MAVS/VISA/Cardif CARD adopts a characteristic six-helix bundle with a Greek-key topology and, in common with a number of other known CARD structures, contains two major polar surfaces on opposite sides of the molecule. One face has a surface-exposed, disordered tryptophan residue that may explain the poor solubility of untagged expression constructs.ConclusionThe IPS-1/MAVS/VISA/Cardif CARD domain adopts the classic CARD fold with an asymmetric surface charge distribution that is typical of CARD domains involved in homotypic protein-protein interactions. The location of the two polar areas on IPS-1/MAVS/VISA/Cardif CARD suggest possible types of associations that this domain makes with the two CARD domains of MDA5 or RIG-I. The N-terminal CARD domains of RIG-I and MDA5 share greatest sequence similarity with IPS-1/MAVS/VISA/Cardif CARD and this has allowed modelling of their structures. These models show a very different charge profile for the equivalent surfaces compared to IPS-1/MAVS/VISA/Cardif CARD.

Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody.

ABSTRACT The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 Å. In the complex, the peptide adopts a β-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alanine-scanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.

Crystal Structure of the Nanb Sialidase from Streptococcus Pneumoniae

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 A resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K(i) of approximately 0.5 mM. In addition, we present the structure to 2.4 A resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K(i) of approximately 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for alpha2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.

Insights Into the Biosynthesis of the Vibrio Cholerae Major Autoinducer Cai-1 from the Crystal Structure of the Plp-Dependent Enzyme Cqsa.

CqsA is an enzyme involved in the biosynthesis of cholerae autoinducer-1 (CAI-1), the major Vibrio cholerae autoinducer engaged in quorum sensing. The amino acid sequence of CqsA suggests that it belongs to the family of alpha-oxoamine synthases that catalyse the condensation of an amino acid to an acyl-CoA substrate. Here we present the apo- and PLP-bound crystal structures of CqsA and confirm that it shares structural homology with the dimeric alpha-oxoamine synthases, including a conserved PLP-binding site. The chemical structure of CAI-1 suggests that decanoyl-CoA may be one substrate of CqsA and that another substrate may be l-threonine or l-2-aminobutyric acid. A crystal structure of CqsA at 1.9-A resolution obtained in the presence of PLP and l-threonine reveals an external aldimine that has lost the l-threonine side chain. Similarly, a 1.9-A-resolution crystal structure of CqsA in the presence of PLP, l-threonine, and decanoyl-CoA shows a trapped external aldimine intermediate, suggesting that the condensation and decarboxylation steps have occurred, again with loss of the l-threonine side chain. It is suggested that this side-chain loss, an observation supported by mass spectrometry, is due to a retro-aldol reaction. Although no structural data have been obtained on CqsA using l-2-aminobutyric acid and decanoyl-CoA as substrates, mass spectrometry confirms the expected product of the enzyme reaction. It is proposed that a region of structure that is disordered in the apo structure is involved in the release of product. While not confirming if CqsA alone is able to synthesize CAI-1, these results suggest possible synthetic routes.

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR.

Background: The Streptococcus pneumoniae sialidase NanC produces a nonspecific inhibitor of hydrolytic sialidases. Results: The NanC crystal structure is presented in complex with mechanistically relevant ligands. Conclusion: A constricted and hydrophobic active site produces 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA) via a covalent intermediate and direct proton abstraction by a catalytic aspartic acid. Significance: Insights into an unusual reaction mechanism will aid the design of sialidase inhibitors. Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.

The structure of Sulfolobus solfataricus 2-keto-3-deoxygluconate kinase

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and utilizes an unusual promiscuous nonphosphorylative Entner-Doudoroff pathway to metabolize both glucose and galactose. It has been proposed that a part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus, in which the 2-keto-3-deoxygluconate kinase (KDGK) is promiscuous for both glucose and galactose metabolism. Recombinant S. solfataricus KDGK protein was expressed in Escherichia coli, purified and crystallized in 0.1 M sodium acetate pH 4.1 and 1.4 M NaCl. The crystal structure of apo S. solfataricus KDGK was solved by molecular replacement to a resolution of 2.0 A and a ternary complex with 2-keto-3-deoxygluconate (KDGlu) and an ATP analogue was resolved at 2.1 A. The complex suggests that the structural basis for the enzymes ability to phosphorylate KDGlu and 2-keto-3-deoxygalactonate (KDGal) is derived from a subtle repositioning of residues that are conserved in homologous nonpromiscuous kinases.

Formate–nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives

The formate-nitrite transporter family is composed of integral membrane proteins that possess six to eight alpha-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to >95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly alpha-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer.

PRIMA-1 Met suppresses colorectal cancer independent of p53 by targeting MEK

PRIMA-1Met is the methylated PRIMA-1 (p53 reactivation and induction of massive apoptosis) and could restore tumor suppressor function of mutant p53 and induce p53 dependent apoptosis in cancer cells harboring mutant p53. However, p53 independent activity of PRIMA-1Met remains elusive. Here we reported that PRIMA-1Met attenuated colorectal cancer cell growth irrespective of p53 status. Kinase profiling revealed that mitogen-activated or extracellular signal-related protein kinase (MEK) might be a potential target of PRIMA-1Met. Pull-down binding and ATP competitive assay showed that PRIMA-1Met directly bound MEK in vitro and in cells. Furthermore, the direct binding sites of PRIMA-1Met were explored by using a computational docking model. Treatment of colorectal cancer cells with PRIMA-1Met inhibited p53-independent phosphorylation of MEK, which in turn impaired anchorage-independent cell growth in vitro. Moreover, PRIMA-1Met suppressed colorectal cancer growth in xenograft mouse model by inhibiting MEK1 activity. Taken together, our findings demonstrate a novel p53-independent activity of PRIMA-1Met to inhibit MEK and suppress colorectal cancer growth.

Crystal structure of VC1805, a conserved hypothetical protein from a Vibrio cholerae pathogenicity island, reveals homology to human p32

The severe diarrhoeal disease cholera is caused by the gram-negative bacterium Vibrio cholerae and continues to be a major cause of morbidity and mortality. Like many Vibrio species V. cholerae inhabits an aquatic ecosystem, and most V. cholerae isolates do not possess the ability to cause cholera. Of more than 200 known serotypes, only O1 and O139 serogroups are highly pathogenic and acknowledged to cause epidemic disease.1 The O1 serogroup can be divided into two groups: classical and El Tor, with the first cholera pandemic, beginning in Asia in 1817, and the subsequent five pandemics probably caused by the classical biotype. The seventh and present pandemic began in 1961 caused by the El Tor biotype.2 In 1992 a novel O-serogroup, O-139, emerged to cause epidemic cholera.3 All V. cholerae O1 and O139 serogroups encode the major virulence factors cholera toxin and toxin coregulated pilus, the latter being encoded on a pathogenicity island, named Vibrio Pathogenicity Island-I (VPI-1).4 Several other genomic regions have been identified that occur mainly among epidemic O1 and O139 serogroup isolates, including the so-called Vibrio seventh pandemic island-I (VSP-I) encoding ORFs VC0175 to VC0185, VSP-II encoding ORFs VC0490 to VC0516 and VPI-2 encoding ORFs VC1758 to VC1809.5–7 A microarray analysis of V. cholerae El Tor isolates was used to identify VSP-I and VSP-II encompassing genes that are possibly responsible for the unique characteristics of the seventh pandemic (El Tor) strains.5 An evolutionary genetic analysis of clinical and environmental isolates suggested that pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.8 VPI-2 is a 57.3 kb region which consists of 52 ORFs present in all toxigenic O1 and O139 serogroup isolates, but lacking in non-O1 and non-O139 nontoxigenic isolates.6 VPI-2 encodes a type-I restriction modification system, a nan-nag region of genes involved in sialic acid metabolism that may play a nutritional role,9 a sialidase/neuraminidase known to convert intestinal higher-order gangliosides to GM1,10 and a region with homology to Mu phage. In addition, VPI-2 contains several genes that code for hypothetical proteins. One of these proteins, VC1805, is a 148 amino acid protein with no function revealed so far through sequence analysis. VC1805 is also encoded in the reduced 20 kb VPI-2 region present among most O139 serogroup isolates that are missing ORFs VC1761 to VC1788.6 A paralogue of VC1805 exists within VSP-II, VC0508 a 147 residue protein that shares 59% amino acid sequence identity with VC1805. A search of the sequence database using PSI-BLAST reveals homologues of VC1805 in several Vibrio species: V. vulnificus, V. splendidus, V. alginolyticus and V. fischeri, and orthologues in Altermonas macleodii, Aeromonas hydrophilia, and some Shewanella species. In addition, PSI-BLAST reveals that the adjacent hypothetical protein VC1804, with 104 residues, is a homologue of VC1805 sharing 26% sequence identity. Similarly the hypothetical protein VC0509 is a homologue of the adjacent protein VC0508. The four proteins VC0508, VC0509, VC1804, and VC1805 are therefore likely to share the same protein fold and be functionally related. As part of a structural genomics approach to understanding the function of hypothetical proteins within V. cholerae genomic islands we have determined the crystal structure of VC1805 to a resolution of 2.1 A using heavy atom isomorphous replacement. The structure reveals a similarity to the human mitochondrial protein p32 that is known to have several binding partners, including the human complement system protein C1q. This study shows that VC1805 does bind C1q, suggesting potential biological roles for the protein and its homologues.