Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae.
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 alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.
Müller
Axel
A
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom.
Severi
Emmanuele
E
Department of Biology, University of York, York YO10 5YW, United Kingdom.
Mulligan
Christopher
C
Department of Biology, University of York, York YO10 5YW, United Kingdom.
Watts
Andrew G
AG
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom.
Kelly
David J
DJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom.
Wilson
Keith S
KS
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom.
Wilkinson
Anthony J
AJ
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom. Electronic address: ajw@ysbl.york.ac.uk.
Thomas
Gavin H
GH
Department of Biology, University of York, York YO10 5YW, United Kingdom. Electronic address: ght2@york.ac.uk.
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D006193:Haemophilus influenzae / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives / Q000737:chemistry*; D027361:Organic Anion Transporters / Q000737:chemistry*; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry*; D037521:Virulence Factors / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D027981:Symporters; D037521:Virulence Factors; C421224:sialic acid transport proteins; D019158:N-Acetylneuraminic Acid