Structural analysis of SulP/SLC26 anion transporters

Content Provider	Semantic Scholar
Author	Bonetto, Greta
Copyright Year	2013
Abstract	The subject of this thesis is a family of anion transporters known as SulP/SLC26 (Sulfate Permease/Solute Carrier 26) family, a large and ubiquitous family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions, whose members were found in eubacteria, plants, fungi, and mammals. The clinical relevance of the SulP/SLC26 gene family has been highlighted with the identification of pathogenic mutations related to hereditary genetic human diseases with diverse symptoms that arise as a result of the different substrate specificities and tissue localizations of the different transporters, such as dystrophic dysplasia (SLC26A2), congenital chloride diarrhoea (SLC26A3) and Pendred syndrome (SLC26A4). The SulP/SLC26 family belongs to the APC (Amino Acid-Polyamine-Organocation) superfamily, one of the largest superfamily of secondary carriers. While some members of other families of the APC superfamily have been structurally characterized, very little is known about the molecular organization of the SulP/SLC26 proteins and no high-resolution three-dimensional structure of full-length sequences is available. The SulP/SLC26 anion transporters share a common structural organization: a highly conserved transmembrane domain and a less conserved cytoplasmic C-terminal portion mainly composed of a STAS domain. The name STAS (Sulfate Transporter and Anti-Sigma factor antagonist) is due to a remote but statistically significant sequence similarity with bacterial ASA (Anti-Sigma factor Antagonist) proteins (Aravind and Koonin, 2000). The bacterial ASA proteins are functionally and structurally well characterized in their 3D structure both by NMR spectroscopy and X-ray crystallography. Unlike these proteins, the STAS domains present in anion transporters are poorly characterized in terms of both their function and structure. Despite the fact that their precise role is unclear, the STAS domains play a fundamental role in the function/regulation of SulP/SLC26 anion transporters. In particular, it has been proposed that the STAS domain, like ASA proteins, could have a role in protein/protein interaction; for instance the STAS domains of SCL26A3, -A4, -A6 and -A9 interact with the R domain of CFTR (Cystic Fibrosis Transmembrane conductance Regulator), the transmembrane protein involved in cystic fibrosis disease. So far three 3D structures of STAS domains from different species are available in literature, two from bacteria and one from mammalian, the latter solved during my Master Degree Thesis in the same laboratory where I've attended the PhD. The structural characterization of the full-length SulP/SLC26 transporters and of their STAS domains is fundamental for the comprehension of their mode of action and it is an essential step for the understanding of the functional consequences of the mutations responsible for related pathologies. To address this issue, one part of my PhD project focused on the production and the structural characterization of STAS domains from different species, and mutants of the STAS domain whose 3D structure have been solved, in order to study the anion-binding site and the possible role of the STAS domain in the transport. We identified a fundamental residue for the proper function of the transporter, probably implicated in the anion translocation within the transmembrane domain. The other part of the project dealt with the production of a selection of full-length SulP/SLC26 transporters from different orthologs, both Prokaryotes and Eukaryotes. To this aim, in collaboration with Prof. Frank Bernhard at the Johann Wolfgang Goethe University of Frankfurt (Germany), I used the cell-free (CF) expression method, an emerging technique for the large-scale production of membrane proteins for structural studies. Sample properties after post-translational solubilization have been analyzed by evaluation of homogeneity and protein stability. This is the first quality evaluation of the SulP/SLC26 transporters produced by CF expression mode in quantities appropriate for structural approaches.
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