Protein Crystallography

Dr P J Baker

Career History

2010 – Present: Senior Lecturer, Dept of Molecular Biology and Biotechnology, The University of Sheffield
2010: Visiting Lecturer, Dept Bioquimica, Faculdad Sciencias, The University of Alicante, Spain
2008 -2010: Lecturer, Dept. of Molecular Biology and Biotechnology, The University of Sheffield
1993 -2010: Senior Experimental Officer (X-ray facility manager) in the Krebs Institute, University of Sheffield
1987-1993: Postdoctoral Research Associate, Dept. of Biochemistry, The University of Sheffield
1984-1987: PhD. Student, Dept. of Biochemistry, The University of Sheffield
1982-1984: Research Assistant, Dept. of Clinical Haematology, University College, London.

In my group we use X-ray crystallography to determine the atomic structure of biological macromolecules and thus to elucidate their structure/function relationships. We work closely with the other structural biology groups in Sheffield and have strong collaborations with numerous research groups across the world. Our research can be divided into three broad areas:

developing new antimicrobial targets
substrate specificity and chiral synthesis in enzymes
the molecular basis of extreme stability in proteins from extremophiles

Developing new antimicrobial targets - structural genomics.

Bacterial pathogens are becoming of increasing importance in health care due to the spread of multi-antibiotic resistant strains. In order to develop new treatment regimes for infectious diseases it is crucial to identify novel therapeutic targets. Essential gene products are ideal, as they are required for cellular viability. In a pilot structural genomics project on Bacillus subtilis, around 30 novel putative essential genes have been identified via functional genomics. Many of these are present in a wide range of bacterial pathogens. The structures of seven of these essential gene products have been determined, one of these is the protein Luxs, which is involved in the regulation of gene expression in response to changes in cell density, a process called quorum sensing. LuxS is thought to catalyze the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The structure of LuxS [Ruzheinikov et al (2001)] shows that it is a homodimer with an apparently novel fold based on an eight stranded beta barrel, flanked by six alpha helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The autoinducer-2 signaling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.

Developing new antimicrobial targets - peptide deformylase

In collaboration with British Biotech (Vernalis) we have also been investigating peptide deformylase (PDF), an essential bacterial metalloenzyme, which deformylates the N-formylmethionine of newly synthesized polypeptides and represents a novel target for antimicrobial chemotherapy. The structure of this enzyme in complex with a potent inhibitor of the enzyme, BB-3497 has recently been determined [Clements, et al.] which shows that the inhibitor binds to the protein to mimic the natural peptidic substrate, with the addition of a bidentate metal chelating hydroxamide to coordinate the metal in the active site. BB-3947 has activity against both Gram-positive and Gram-negative bacteria and the mode of action is primarily bacteriostatic. It is rapidly and well absorbed following oral administration and is effective in a mouse systemic infection model with S. aureus, showing that PDF is a valid target for antimicrobial drug development.

Figure 1. The potent inhibitor BB-3497 bound in the active site cleft of peptidedeformylase shown as a cartoon (left) and a surface representation (right).

Substrate specificity and chiral synthesis - the amino acid dehydrogenase family

The use of enzymes as biocatalysts to produce novel, chirally pure chemicals is of increasing industrial importance. Understanding how Nature has evolved different strategies for altering substrate specificity in families of enzymes is crucial in using site directed mutagenesis to produce mutant enzymes acting on novel compounds. Investigating the molecular basis of substrate specificity in the amino acid dehydrogenase family is one area of my research. Determination of the atomic structures of glutamate dehydrogenase, leucine dehydrogenase and phenylalanine dehydrogenase has shown that these three enzymes share the same three dimensional subunit structures, similar active sites, and identical residues involved in the catalytic chemistry. However, a combination of point mutations and subtle main chain movement, in the substrate side chain binding sites linked to the quaternary structure, account for the differential substrate specificity seen in this family of enzymes [Baker et al, Biochemistry (1997)]. A program of site-directed mutagenesis, is now underway, with the object of producing mutant enzymes with novel specificities. These can be used for the production of chirally pure novel non-proteogenic amino acids for use in the pharmaceutical industry and also as diagnostic reagents for the detection of raised levels of amino acids in serum, crucial for the correct diagnosis of a number of genetic diseases of neonates such as phenylketonuria, homocysteinuria and maple syrup urine disease. We have recently succeeded in engineering a member of this enzyme family to have the ideal properties required in a spectrophotometric assay for phenylketonuria and the mutant is undergoing trials in the clinic [Wang et al (2001)].

Substrate specificity and chiral synthesis - methylaspartate ammonia lyase

Another enzyme that has biotechnological potential for the chiral synthesis of a number of novel amino acids is methylaspartate ammonia lyase (MAL), which catalyzes the magnesium-dependent reversible alpha-beta elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 Å crystal structure of the dimeric Citrobacter amalonaticus MAL has been determined using Seleno-methionine MAD experiments and shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel [Levy et al (2002)]. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Figure 2. A comparison of methylaspartate ammonia lyase (left) and enolase (right), PDB code 1EBG, showing the similarity between their three dimensional structures.

Substrate specificity and chiral synthesis - alanine dehydrogenase.

Alanine dehydrogenase (AlaDH), catalyses the NADH-dependent reversible reductive amination of pyruvate to L-alanine and is a key factor in the assimilation of L-alanine as an energy source through the tricarboxylic acid cycle during sporulation. The structure of alanine dehydrogenase from the cyanobacterium Phormidium lapideum and also from Bacillus stearothermophilus [Baker et al (1998)], shows that, despite catalyzing the same oxidative deamination reaction as the other amino acid dehydrogenases, AlaDH is structurally totally different, sharing the same fold as that of the D-2-hydroxyacid dehydrogenases and component dI of transhydrogenase.

Membrane bound ion pumps are involved in metabolic regulation, osmoregulation, cell signalling, nerve transmission and energy transduction. How the ion electrochemical gradient interacts with the scalar chemistry, and how the catalytic machinery is gated to ensure high coupling efficiency, are fundamental to the mechanism of action of such pumps. Transhydrogenase is a conformationally-coupled proton pump linking a proton gradient to the redox reaction between NAD(H) and NADP(H) and is important in the production of NADPH for the biosynthesis of amino acids and steroids, for detoxification, including limitation of the damage caused by free radicals and for the correct poising of the cellular electrochemical gradient. The enzyme has three components, dI binds NAD(H), dII spans the membrane and dIII binds NADP(H). Using seleno-methionine multiwavelength anomalous dispersion data collected at the ESRF synchrotron at Grenoble, with 52 selenium sites in the asymmetric unit, the structure of the dI component of this enzyme from Rhodospirulum rubrum [Buckley et al (2000)] has been successfully determined. This structure has revealed how the dI and dIII polypeptides may associate in the intact complex and explains the gating of the scalar hydride-tr ansfer reaction.

Figure 3. The dimer of transhydrogenase component d1 with bound NAD

The molecular basis of extreme stability in proteins - glutamate dehydrogenase

As part of our research programme on amino acid dehydrogenases, we have determined the structure of glutamate dehydrogenase (GluDH) from Pyrococcus furiosus and Thermococcus litoralis -hyperthermophilic organisms with slightly different thermostabilities. These structures reveal that the formation of extended ion-pair networks is a major stabilizing feature in the adaptation of the organism to life at 100C [Britton et al (1999)]. An extra ion-pair network has been engineered into the T.litoralis enzyme, increasing its thermostability [Vetriani et al (1998)]. The structure determination of other hyperthermophilic proteins, enzymes from psychrophilic sources and proteins from halophiles is now extending this work into understanding the molecular basis of thermostability, cold tolerance and the forces governing the adaptation of proteins to extremely high concentrations of salt.

Figure 4. The hexamer of glutamate dehydrogenase (left) and the salt bridge network of 18 residues (right) that helps to stabilize the hexamer of the hyperthermophilic glutamate dehydrogenase from Pyrococcus furiosus.

The molecular basis of extreme stability in proteins - phosphoglucose isomerase.

We have recently determined the structure of the phosphoglucose isomerase (PGI) from Pyrococcus furiosus [Berrisford et al (2003)]. This PGI is a dimer of identical 23.5-kDa subunits and catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The structure has revealed that the fold of this enzyme is based on a cupin domain and is completely different to the alpha-beta-alpha sandwich of the much larger structure of the eukaryotic and bacterial PGIs. Despite these quite different architectures of the polypeptide backbones, the active site of both classes of PGI are remarkably similar, suggesting that they use similar reaction mechanisms. At its optimum temperature of 90C, PF PGI has a half-life of 2.4 h. Analysis of the structure has shown, like in other thermostable enzymes, a number of ion pair interactions, which cluster into networks. We are currently investigating whether these networks are involved in the thermostability of this this enzyme.

Figure 5. - The dimer of phosphoglucose isomerase from Pyrococcus furiosus.

Collaborating research groups.

Professor S. Foster (Sheffield)
Professor D.W Rice (Sheffield)
Dr. J.Gilmour (Sheffield)
British Biotech (Vernalis)
Professor K. Soda (Kyoto, Japan)
Professor N. Esaki (Kyoto, Japan)
Professor H. Misono (Kochi, Japan)
Professor T. Ohshima (Tokushima, Japan)
Professor Y. Asano (Toyama, Japan)
Professor Y. Sawa (Shimane, Japan)
Professor B. Jackson (Birmingham, U.K)
Professor P.C.Engel (UCD, Dublin)
Professor G. Alfreddson (Rekjavic, Iceland)
Professor J. van der Oost, (Wagenigen, Netherlands)
Professor F. Robb (Maryland, USA)
Professor R. Scandura (Rome, Italy)
Professor G. DiPrisco, (Napoli, Italy)
Professor L.Camardella (Napoli, Italy)
Professor G. Antrinikian (Hamburg, Germany)
Professor P. Forterre (Paris, France)
Professor N. Glansdorff (Brussels, Belgium)
Professor R. Ladenstein (Stockholm, Sweden)
Professor W. Liebl (Gottingen, Germany)

Selected Publications

Abd Aziz, A. G., Sedelnikova, S. E., Ruzheinikov, S. N., Thorpe, S., Mohamed, R., Nathan, S., Rafferty, J. B., Baker, P.J. & Rice, D. W. (2012). Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei. Acta Cryst. F 68, 917-922.

Abd Aziz, A. G., Ruzheinikov, S. N., Sedelnikova, S. E., Mohamed, R., Nathan, S., Baker, P.J. & Rice, D. W. (2012). Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein. Acta Cryst. F 68, 347-350.

Cruz-Migoni, A., Ruzheinikov, S. N., Sedelnikova, S. E., Obeng, B., Chieng, S., Mohamed, R., Nathan, S., Baker, P.J. & Rice, D. W. (2011). Cloning, purification and crystallographic analysis of a hypothetical protein, BPSL1549, from Burkholderia pseudomallei. Acta Cryst. F 67, 1623-1626.

Cruz-Migoni, A., Hautbergue, G. M., Artymiuk, P. J., Baker, P.J., Bokori-Brown, M., Chang, C.-T., Dickman, M. J., Essex-Lopresti, A., Harding, S. V., Mahadi, N. M., Marshall, L. E., Mobbs, G. W., Mohamed, R., Nathan, S., Ngugi, S. A., Ong, C., Ooi, W. F., Partridge, L. J., Phillips, H. L., Raih, M. F., Ruzheinikov, S., Sarkar-Tyson, M., Sedelnikova, S. E., Smither, S. J., Tan, P., Titball, R. W., Wilson, S. A. & Rice, D. W. (2011). A Burkholderia pseudomallei Toxin Inhibits Helicase Activity of Translation Factor eIF4A. Science 334, 821-824.

Tomlinson, J. H., Green, V. L., Baker, P.J. & Williamson, M. P. (2010). Structural origins of pH-dependent chemical shifts in the B1 domain of protein G. Proteins 78, 3000-3016.

Domenech, J., Baker, P.J., Sedelnikova, S. E., Rodgers, H. F., Rice, D. W. & Ferrer, J. (2009). Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei. Acta Cryst. F 65, 415-418.

Baker, P.J., Britton, K. L., Fisher, M., Esclapez, J., Pire, C., Jose Bonete, M., Ferrer, J. & Rice, D. W. (2009). Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily. P.N.A.S. 106, 779-784.

Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P.J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J. & Czaplewski, L. G. (2008). An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 321, 1673-1675.

Xu, L., Sedelnikova, S. E., Baker, P.J., Hunt, A., Errington, J. & Rice, D. W. (2007). Crystal structure of S-aureus YlaN, an essential leucine rich protein involved in the control of cell shape. Proteins 68, 438-445.

Macmaster, R., Sedelnikova, S., Baker, P.J., Bolt, E. L., Lloyd, R. G. & Rafferty, J. B. (2006). RusA Holliday junction resolvase: DNA complex structure - insights into selectivity and specificity. Nuc. Acids Res. 34, 5577-5584.

Britton, K. L., Baker, P.J., Fisher, M., Ruzheinikov, S., Gilmour, D. J., Bonete, M. J., Ferrer, J., Pire, C., Esclapez, J. & Rice, D. W. (2006). Analysis of protein solvent interactions in glucose dehydrogenase from the extreme halophile Haloferax mediterranei. P.N.A.S 103, 4846-4851.

Berrisford, J. M., Hounslow, A. M., Akerboom, J., Hagen, W. R., Brouns, S. J. J., van der Oost, J., Murray, I. A., Blackburn, G. M., Waltho, J. P., Rice, D. W. & Baker, P.J. (2006). Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase. J.Mol.Biol. 358, 1353-1366.

Ruzheinikov, S. N., Taal, M. A., Sedelnikova, S. E., Baker, P.J. & Rice, D. W. (2005). Substrate-induced conformational changes in Bacillus subtilis glutamate racemase and their implications for drug discovery. Structure 13, 1707-1713.

Glynn, S. E., Baker, P.J., Sedelnikova, S. E., Davies, C. L., Eadsforth, T. C., Levy, C. W., Rodgers, H. F., Blackburn, G. M., Hawkes, T. R., Viner, R. & Rice, D. W. (2005). Structure and mechanism of imidazoleglycerol-phosphate dehydratase. Structure 13, 1809-1817.

Ruzheinikov, S. N., Das, S. K., Sedelnikova, S. E., Baker, P.J., Artymiuk, P. J., Garcia-Lara, J., Foster, S. J. & Rice, D. W. (2004). Analysis of the open and closed conformations of the GTP-binding protein YsxC from Bacillus subtilis. J.Mol.Biol. 339, 265-278.

Hartley, A., Glynn, S. E., Barynin, V., Baker, P.J., Sedelnikova, S. E., Verhees, C., de Geus, D., van der Oost, J., Timson, D. J., Reece, R. J. & Rice, D. W. (2004). Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase. J.Mol.Biol. 337, 387-398.

Berrisford, J. M., Akerboom, J., Brouns, S., Sedelnikova, S. E., Turnbull, A. P., van der Oost, J., Salmon, L., Hardre, R., Murray, I. A., Blackburn, G. M., Rice, D. W. & Baker, P.J. (2004). The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis. J.Mol.Biol. 343, 649-657.

Baker, P.J. (2004). From hyperthermophiles to psychrophiles: the structural basis of temperature stability of the amino acid dehydrogenases. Biochem. Soc. Trans 32, 264-268.

Rafferty, J. B., Bolt, E. L., Muranova, T. A., Sedelnikova, S. E., Leonard, P., Pasquo, A., Baker, P.J., Rice, D. W., Sharpies, G. J. & Lloyd, R. G. (2003). The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold. Structure 11, 1557-1567.

Irwin, J. A., Lynch, S. V., Coughlan, S., Baker, P.J., Gudmundsson, H. M., Alfredsson, G. A., Rice, D. W. & Engel, P. C. (2003). Alanine dehydrogenase from the psychrophilic bacterium strain PA-43: overexpression, molecular characterization, and sequence analysis. Extremophiles 7, 135-143.

Dall'Antonia, F., Baker, P.J. & Schneider, T. R. (2003). Optimization of selenium substructures as obtained from SHELXD. Acta Cryst. D 59, 1987-1994.

Berrisford, J. M., Akerboom, J., Turnbull, A. P., de Geus, D., Sedelnikova, S. E., Staton, I., McLeod, C. W., Verhees, C. H., van der Oost, J., Rice, D. W. & Baker, P.J. (2003). Crystal structure of Pyrococcus furiosus phosphoglucose isomerase - Implications for substrate binding and catalysis. J.Biol.Chem. 278, 33290-33297.

Roujeinikova, A., Baldock, C., Simon, W. J., Gilroy, J., Baker, P.J., Stuitje, A. R., Rice, D. W., Slabas, A. R. & Rafferty, J. B. (2002). X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site. Structure 10, 825-835.

Levy, C. W., Buckley, P. A., Sedelnikova, S., Kato, Y., Asano, Y., Rice, D. W. & Baker, P.J. (2002). Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase. Structure 10, 105-113.

Ruzheinikov, S. N., Das, S. K., Sedelnikova, S. E., Hartley, A., Foster, S. J., Horsburgh, M. J., Cox, A. G., McCleod, C. W., Mekhalfia, A., Blackburn, G. M., Rice, D. W. & Baker, P.J. (2001). The 1.2 angstrom structure of a novel quorum-sensing protein, Bacillus subtilis LuxS. J.Mol.Biol 313, 111-122.

Ruzheinikov, S. N., Burke, J., Sedelnikova, S., Baker, P.J., Taylor, R., Bullough, P. A., Muir, N. M., Gore, M. G. & Rice, D. W. (2001). Glycerol dehydrogenase: Structure, specificity, and mechanism of a family III polyol dehydrogenase. Structure 9, 789-802.

Clements, J. M., Beckett, R. P., Brown, A., Catlin, G., Lobell, M., Palan, S., Thomas, W., Whittaker, M., Wood, S., Salama, S., Baker, P.J., Rodgers, H. F., Barynin, V., Rice, D. W. & Hunter, M. G. (2001). Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor. Antimicrobial Agents and Chemotherapy 45, 563-570.

Buckley, P. A., Jackson, J. B., Schneider, T., White, S. A., Rice, D. W. & Baker, P.J. (2000). Protein-protein recognition, hydride transfer and proton pumping in the transhydrogenase complex. Structure 8, 809-815.

Baker, P.J., Sawa, Y., Shibata, H., Sedelnikova, S. E. & Rice, D. W. (1998). Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase. Nature Struc. Biol. 5, 561-567.