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J. Biol. Chem., Vol. 281, Issue 31, 22212-22222, August 4, 2006

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Conservation of Structure and Mechanism in Primary and Secondary Transporters Exemplified by SiaP, a Sialic Acid Binding Virulence Factor from Haemophilus influenzae^*

Axel Müller, Emmanuele Severi, Christopher Mulligan, Andrew G. Watts¹, David J. Kelly^¶, Keith S. Wilson, Anthony J. Wilkinson², and Gavin H. Thomas³

From the Structural Biology Laboratory, Department of Chemistry and Department of Biology, University of York, York YO10 5YW, United Kingdom and ^¶Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom

Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an -helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.

Received for publication, April 11, 2006 , and in revised form, May 11, 2006.

The atomic coordinates and structure factors (code 2CEY (unliganded structure) and 2CEX (Neu5Ac2en structure)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The work described here was funded by the European Commission as Structural Proteomics in Europe (SPINE), Contract QLG2-CT-2002-00988 under the Research Technological Development (RTD) program "Quality of Life and Management of Living Resources" and by a grant from the United Kingdom Biotechnology and Biological Science Research Council (to G. H. T. and D. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Dept. of Pharmacy and Pharmacology, University of Bath, BA2 7AY, UK.

² To whom correspondence may be addressed. Tel.: 44-1904-328261; E-mail: ajw{at}ysbl.york.ac.uk.

³ To whom correspondence may be addressed. Tel.: 44-1904-328268; E-mail: ght2{at}york.ac.uk.

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