Abstract
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.
Highlights
Feature of uptake systems, including the well studied ATP binding cassette (ABC)4 transporters, is an extracytoplasmic solute receptor (ESR), which captures the substrate for the transporter and delivers it to the membrane subunits [1]
Unusual features of tripartite ATP-independent periplasmic (TRAP) ESR proteins reported in the literature has come from kinetic data that suggest the protein predominates in a closed conformation even in the absence of ligand [33]
This is different from all other ESR proteins for which binding data are available, implying a mechanistic difference between TRAP ESR proteins and ABC ESR proteins
Summary
Expression and Purification of SiaP—The SiaP protein was purified from E. coli using a modification of the methods described in Severi et al [13]. The breakthrough came via the use of the experimental phase probability distributions in terms of the Hendrickson-Lattman coefficients as restraints during the ARP/wARP-REFMAC rebuilding giving a model with more than 570 of the expected 612 residues and with most of the side chain correctly assigned This model was used for molecular replacement with the Form 2 native data, and MOLREP [23] subsequently provided an essentially complete model using ARP/wARP-REFMAC. The Form 2 crystal structure was in turn used as a search model in molecular replacement calculations with the Form 3 crystal data in the program MOLREP, leading to the identification of four molecules in the asymmetric unit. Steady-state and Stopped-flow Fluorescence Spectroscopy— Steady-state protein fluorescence studies were performed as previously described [13] unless outlined in the AUGUST 4, 2006 VOLUME 281 NUMBER 31
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