Publications (13)50.19 Total impact
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Article: Towards a Hepatitis C Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody.
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ABSTRACT: The E2 envelope glycoprotein of 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 has been determined to 1.8Å. 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.Journal of Virology 09/2012; · 5.40 Impact Factor -
Article: Mechanism of recombination of the P+H(A)- radical pair in mutant Rhodobacter sphaeroides reaction centers with modified free energy gaps between P+B(A)- and P+H(A)-.
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ABSTRACT: The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.The Journal of Physical Chemistry B 11/2011; 115(44):13037-50. · 3.70 Impact Factor -
Article: Formate-nitrite transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives.
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ABSTRACT: 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.Protein Expression and Purification 12/2009; 71(2):184-9. · 1.59 Impact Factor -
Article: Insights into the Biosynthesis of the Vibrio cholerae Major Autoinducer CAI-1 from the Crystal Structure of the PLP-Dependent Enzyme CqsA
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ABSTRACT: 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 α-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 α-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-Å 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-Å-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.Journal of Molecular Biology 08/2009; · 4.00 Impact Factor -
Article: The structure of Sulfolobus solfataricus 2-keto-3-deoxygluconate kinase.
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ABSTRACT: 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 enzyme's 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.Acta crystallographica. Section D, Biological crystallography 01/2009; 64(Pt 12):1283-7. · 12.67 Impact Factor -
Article: Crystal structure of the NanB sialidase from Streptococcus pneumoniae.
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ABSTRACT: 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.Journal of Molecular Biology 10/2008; 384(2):436-49. · 4.00 Impact Factor -
Article: Crystal structure of VC1805, a conserved hypothetical protein from a Vibrio cholerae pathogenicity island, reveals homology to human p32.
Proteins Structure Function and Bioinformatics 06/2008; 71(3):1563-71. · 3.39 Impact Factor -
Article: The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.
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ABSTRACT: 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 0.97A 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 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A 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.Journal of Biological Chemistry 05/2008; 283(14):9080-8. · 4.77 Impact Factor -
Article: Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain.
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ABSTRACT: IPS-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. The crystal structure of human IPS-1/MAVS/VISA/Cardif CARD has been determined to 2.1A 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. The 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.BMC Structural Biology 02/2008; 8:11. · 2.48 Impact Factor -
Article: Structural responses to cavity-creating mutations in an integral membrane protein.
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ABSTRACT: X-ray crystallography has been used to investigate the extent of structural changes in mutants of the purple bacterial reaction center that assemble without a particular ubiquinone or bacteriopheophytin cofactor. In the case of the bacteriopheophytin-exclusion mutant, in which Ala M149 was replaced by Trp (AM149W), the quality of protein crystals was improved over that seen in previous work by minimizing illumination, time, and temperature during the purification protocol and carrying out crystal growth at 4 degrees C after overnight incubation at 18 degrees C. The X-ray crystal structure of the AM149W mutant, determined to a resolution of 2.2 A, showed very little change in protein structure despite the absence of the bacteriopheophytin cofactor. Changes in the electron density map in the region of the cofactor binding site could be accounted for by changes in the conformation of the phytol side chains of adjacent cofactors and the presence of a buried water molecule. Residues lining the vacated binding pocket did not show any significant changes in conformation or increases in disorder as assessed through crystallographic atomic displacement parameters (B-factors). The X-ray crystal structure of a reaction center lacking the primary acceptor ubiquinone through mutation of Ala M248 to Trp (AM248W) was also determined, to a resolution of 2.8 A. Again, despite the absence of an internal cofactor only very minor changes in protein structure were observed. This is in contrast to a previous report on a reaction center lacking this ubiquinone through mutation of Ala M260 to Trp (AM260W) where more extensive changes in structure were apparent. All three mutant reaction centers showed a decrease in thermal stability when housed in the native membrane, but this decrease was smaller for the AM260W mutant than the AM248W complex, possibly due to beneficial effects of the observed changes in protein structure. The lack of major changes in protein structure despite the absence of large internal cofactors is discussed in terms of protein rigidity, the protective influence of the adaptable membrane environment, and the role of small molecules and ions as packing material in the internal cavities created by this type of mutation.Biochemistry 10/2007; 46(37):10461-72. · 3.42 Impact Factor -
Article: Strong effects of an individual water molecule on the rate of light-driven charge separation in the Rhodobacter sphaeroides reaction center.
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ABSTRACT: The role of a water molecule (water A) located between the primary electron donor (P) and first electron acceptor bacteriochlorophyll (B(A)) in the purple bacterial reaction center was investigated by mutation of glycine M203 to leucine (GM203L). The x-ray crystal structure of the GM203L reaction center shows that the new leucine residue packs in such a way that water A is sterically excluded from the complex, but the structure of the protein-cofactor system around the mutation site is largely undisturbed. The results of absorbance and resonance Raman spectroscopy were consistent with either the removal of a hydrogen bond interaction between water A and the keto carbonyl group of B(A) or a change in the local electrostatic environment of this carbonyl group. Similarities in the spectroscopic properties and x-ray crystal structures of reaction centers with leucine and aspartic acid mutations at the M203 position suggested that the effects of a glycine to aspartic acid substitution at the M203 position can also be explained by steric exclusion of water A. In the GM203L mutant, loss of water A was accompanied by an approximately 8-fold slowing of the rate of decay of the primary donor excited state, indicating that the presence of water A is important for optimization of the rate of primary electron transfer. Possible functions of this water molecule are discussed, including a switching role in which the redox potential of the B(A) acceptor is rapidly modulated in response to oxidation of the primary electron donor.Journal of Biological Chemistry 08/2005; 280(29):27155-64. · 4.77 Impact Factor -
Article: Primary charge separation between P* and BA: Electron-transfer pathways in native and mutant GM203L bacterial reaction centers
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ABSTRACT: Coherent components in the dynamics of decay of stimulated emission from the primary electron donor excited state P*, and of population of the product charge-separated states and , were studied in GM203L mutant reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides by measuring oscillations in the kinetics of absorbance changes at 940 nm (P* stimulated emission region), 1020 nm ( absorption region) and 760 nm (HA bleaching region). Absorbance changes were induced by excitation of P (870 nm) with 18 fs pulses at 90 K. In the GM203L mutant, replacement of Gly M203 by Leu results in exclusion of the crystallographically defined water molecule (HOH55) located close to the oxygen of the 131-keto carbonyl group of BA and to His M202, which provides the axial ligand to the Mg of the PB bacteriochlorophyll. The results of femtosecond measurements were compared with those obtained with Rb. sphaeroides R-26 RCs containing an intact water HOH55. The main consequences of the GM203L mutation were found to be as follows: (i) a low-frequency oscillation at 32 cm−1, which is characteristic of the HOH55-containing RCs, disappears from the kinetics of absorbance changes at 1020 and 760 nm in the mutant RC; (ii) electron transfer from P* to BA in the wild type RC was characterized by two time constants of 1.1 ps (80%) and 4.3 ps (20%), but in the GM203L mutant was characterized by a single time constant of 4.3 ps, demonstrating a slowing of primary charge separation. The previously postulated rotation of water HOH55 with a fundamental frequency of 32 cm−1, triggered by electron transfer from P* to BA, was confirmed by observation of an isotopic shift of the 32 cm−1 oscillation in the kinetics of population in deuterated, pheophytin-modified RCs of Rb. sphaeroides R-26, by a factor of 1.6. These data are discussed in terms of the influence of water HOH55 on the energetics of the reaction, and protein dynamic events that occur on the time scale of this reaction.Chemical Physics. -
Article: Formate–nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives
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ABSTRACT: The formate–nitrite transporter family is composed of integral membrane proteins that possess six to eight α-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 α-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.Protein Expression and Purification.
Top co-authors
Top Journals
Institutions
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2008–2012
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University of St Andrews
- Centre for Biomolecular Sciences
Saint Andrews, SCT, United Kingdom
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2011
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Uniwersytet im. Adama Mickiewicza w Poznaniu
Poznań, Greater Poland Voivodeship, Poland
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2005–2007
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University of Bristol
- School of Biochemistry
Bristol, ENG, United Kingdom
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