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Research Article

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding

¹ Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom
² Arrow Therapeutics, London SE1 1DA, United Kingdom
³ Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom

(RECEIVED November 29, 2005; FINAL REVISION January 10, 2006; ACCEPTED January 12, 2006)

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularlythrough hospital acquired infection. The spread ofMRSAmeans that novel targets are required to developpotential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase(TMK) is attractive as an antibacterial target as it is essential for providing components forDNAsynthesis.Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylatekinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and showvariations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition toopen and closed forms of SaTMK, an intermediate conformation inTMP binding is observed, inwhich thesite is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, withhelix 3 shifting position initially, followed by movement of 2 to close the substrate site. In addition, weobserve significant conformational differences in the TMP-binding site in SaTMK as compared toavailable TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosisas well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to thethymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMKstructural differences mean that design of compounds highly specific for the S. aureus enzyme lookspossible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.

Keywords: S. aureus; thymidylate kinase; X-ray crystallography; substrate-induced conformational change; drug design

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052002406

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