Low Resolution Structure of a Bacterial SLC26 Transporter Reveals Dimeric Stoichiometry and Mobile Intracellular Domains

Frank Gabel,Arnaud Javelle,Eleni Karinou,James H Naismith,Emma L.R Compton

doi:10.1074/jbc.m111.244533

Abstract

The SLC26/SulP (solute carrier/sulfate transporter) proteins are a superfamily of anion transporters conserved from bacteria to man, of which four have been identified in human diseases. Proteins within the SLC26/SulP family exhibit a wide variety of functions, transporting anions from halides to carboxylic acids. The proteins comprise a transmembrane domain containing between 10-12 transmembrane helices followed a by C-terminal cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domain. These proteins are expected to undergo conformational changes during the transport cycle; however, structural information for this family remains sparse, particularly for the full-length proteins. To address this issue, we conducted an expression and detergent screen on bacterial Slc26 proteins. The screen identified a Yersinia enterocolitica Slc26A protein as the ideal candidate for further structural studies as it can be purified to homogeneity. Partial proteolysis, co-purification, and analytical size exclusion chromatography demonstrate that the protein purifies as stable oligomers. Using small angle neutron scattering combined with contrast variation, we have determined the first low resolution structure of a bacterial Slc26 protein without spectral contribution from the detergent. The structure confirms that the protein forms a dimer stabilized via its transmembrane core; the cytoplasmic STAS domain projects away from the transmembrane domain and is not involved in dimerization. Supported by additional biochemical data, the structure suggests that large movements of the STAS domain underlie the conformational changes that occur during transport.

Highlights

SLC26 transporters must undergo sequential conformational changes during the transport cycle as typified by prestin (SLC26A5), the cochlear protein that enables animals to efficiently detect high frequency sounds [11, 12]
YeSlc26A2 Purifies as Stable Oligomer—Mammalian integral membrane proteins are notoriously difficult to handle; studies on prokaryotic homologues have proven crucial in characterizing the structure and function of their human counterparts [21]
A subsequent expression and detergent screen identified the Y. enterocolitica protein YP001005307.1 as the ideal candidate for further structural studies as it can be purified to homogeneity in the detergent Fos-choline-12 and elutes as a single, stable and symmetrical peak from size exclusion chromatography (Fig. 2A)

Summary

Introduction

SLC26 transporters must undergo sequential conformational changes during the transport cycle as typified by prestin (SLC26A5), the cochlear protein that enables animals to efficiently detect high frequency sounds [11, 12]. Characterizing the architecture of SLC26 transporters is of particular interest, paving the way to an understanding of their domain organization, structure, and associated conformational changes. Low Resolution Structure of a Bacterial SLC26A Protein gent and obtain a low resolution model of a purified SLC26 protein alone in solution, giving the first insight into the domain organization of this important protein family. Our low resolution structure, combined with other structural and biochemical information, provides a structural model for the conformational changes associated with the transport cycle in SLC26/SulP proteins

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Conclusion