The STAS domain — a link between anion transporters and antisigma-factor antagonists

Anion transporters of the sulfate transporter family are of major interest, as their malfunction is implicated in three human diseases: diastrophic dysplasia/achondrogenesis type IB (DTD) [1Hastbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve-Daly M.P. Daly M. et al.The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 2Superti-Furga A. Hastbacka J. Wilcox W.R. Cohn D.H. van der Harten H.J. Rossi A. Blau N. et al.Achondrogenesis type IB is caused by mutations in the diastrophic dysplasia sulphate transporter gene.Nat Genet. 1996; 12: 100-102Crossref PubMed Scopus (178) Google Scholar], Pendred’s syndrome (PDS) [3Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. et al.Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).Nat Genet. 1997; 17: 411-422Crossref PubMed Scopus (948) Google Scholar] and congenital chloride diarrhoea (CLD) [4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. et al.Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea.Nat Genet. 1996; 14: 316-319Crossref PubMed Scopus (322) Google Scholar]. The CLD gene is also downregulated in intestinal adenomas and adenocarcinomas [5Schweinfest C.W. Henderson K.W. Suster S. Kondoh N. Papas T.S. Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.Proc Natl Acad Sci USA. 1993; 90: 4166-4170Crossref PubMed Scopus (181) Google Scholar]. The products of these genes are distinct but related anion transporters that contain 12 trans-membrane helices followed by a cytoplasmic domain at the carboxyl terminus. The DTD gene encodes a sulfate transporter [6Satoh H. Susaki M. Shukunami C. Iyama K. Negoro X. Hiraki Y. Functional analysis of diastrophic dysplasia sulfate transporter.Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans. J Biol Chem. 1998; 273: 12307-12315Google Scholar], the PDS gene a potential iodide-chloride transporter [7Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karnishki L.P. The Pendred syndrome gene encodes a chloride-iodide transport protein.Nat Genet. 1999; 21: 440-443Crossref PubMed Scopus (497) Google Scholar] and the CLD gene a chloride–NaHCO3− exchanger [8Melvin J.E. Park K. Richardson L. Schultheis P.J. Shull G.E. Mouse down-regulated in adenoma (DRA) is an intestinal Cl(−)/HCO(3)(−) exchanger and is up-regulated in colon of mice lacking the NHE3 Na(+)/H(+) exchanger.J Biol Chem. 1999; 274: 22855-22861Crossref PubMed Scopus (236) Google Scholar]. Related transporters with the same domain organization, mainly involved in sulfate transport, are present in other eukaryotes and in many bacteria [9Smith F.W. Ealing P.M. Hawkesford M.J. Clarkson D.T. Plant members of a family of sulfate transporters reveal functional subtypes.Proc Natl Acad Sci USA. 1995; 92: 9373-9377Crossref PubMed Scopus (267) Google Scholar]. We describe here an unexpected, statistically significant similarity between the carboxy-terminal cytoplasmic domains of these transporters and the bacterial antisigma-factor antagonists (ASA) typified by Bacillus subtilis SPOIIAA. In a PSI-BLAST search [10Altschul S.F. Koonin E.V.P. PSI-BLAST — a tool for making discoveries in sequence databases.Trends Biochem Sci. 1998; 23: 444-447Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 11Altschul S. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (56915) Google Scholar] seeded with the SPOIIAA sequence from Bacillus stearothermophilus, with a profile inclusion cut-off of 0.01, the carboxy-terminal domain of the human disease-associated transporters and their eukaryotic and bacterial homologs were detected with random expectation (E) values of 10−3–10−4 within five iterations. In reciprocal searches initiated with the CLD transporter carboxy-terminal domain, bacterial ASAs were detected with E values of 10−4–10−6 in the third iteration. The nuclear magnetic resonance structure of the ASA SPOIIAA [12Kovacs H. Comfort D. Lord M. Campbell I.D. Yudkin M.D. Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor sigmaF of Bacillus subtilis.Proc Natl Acad Sci USA. 1998; 95: 5067-5071Crossref PubMed Scopus (55) Google Scholar] provides the structural framework for the emerging domain superfamily. A multiple alignment of this superfamily was constructed using the CLUSTALW program [13Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (53470) Google Scholar] and adjusted using the PSI-BLAST results (Figure 1). The alignment of the carboxy-terminal domains of anion transporters was used for secondary structure prediction-based threading through the Protein Databank (PDB) database using the PHD-TOPITS program [14Rost B. Schneider R. Sander C. Protein fold recognition by prediction-based threading.J Mol Biol. 1997; 270: 471-480Crossref PubMed Scopus (219) Google Scholar]. The best hit was the PDB entry for SPOIIAA (PDB code 1AUZ), with a Z-score of 3.1, which strongly suggests a structure similar to that of SPOIIAA. Thus, ASAs and the cytoplasmic portions of anion transporters define a previously undetected, ancient, conserved domain that we named STAS after sulfate transporters and antisigma-factor antagonists. By mapping the conserved motifs apparent from the multiple alignment (Figure 1) onto the SPOIIAA structure (Figure 2), the conservation was traced largely to the four strands that form the scaffold of the STAS domain. In addition, the turn between the two amino-terminal strands and the long loop between strand 3 and helix 2 are strongly conserved and inserts appear not to be tolerated in these elements (Figure 1, Figure 2). Most of the variability is in the loop between helix 1 and strand 3, with α-helical inserts of considerable size seen in some of the anion transporters (Figure 1). A comparison of the alignment with the tertiary structure shows that the carboxy-terminal region of the STAS domain forms a characteristic α-helical handle-like structure (Figure 1, Figure 2). The identification of the STAS domain in the ASAs and the anion transporters provides functional clues for the regulation of anion transport. ASA-like proteins have been found in a variety of bacteria, such as Gram-positive bacteria, Actinomycetes, Cyanobacteria, chlamydiae, Treponema and Thermotoga. These proteins positively regulate sigma factors by interacting with the anti-sigma factor — a protein kinase. This kinase, in turn, phosphorylates the ASA on a serine in the conserved loop (Figure 2) and thus inactivates it; the ASA can be re-activated through dephosphorylation by a phosphatase [15Duncan L. Alper S. Losick R. SpoIIAA governs the release of the cell-type specific transcription factor sigma F from its anti-sigma factor SpoIIAB.J Mol Biol. 1996; 260: 147-164Crossref PubMed Scopus (96) Google Scholar]. Thus, the STAS domain is at the center of protein–protein interactions in the sigma-factor regulation network. It has been shown that SPOIIAA binds GTP and ATP and possesses a weak NTPase activity that is abolished by phosphorylation or by mutation of the phosphorylatable serine in the conserved loop (Figure 1) [16Najafi S.M. Harris D.A. Yudkin M.D. The SpoIIAA protein of Bacillus subtilis has GTP-binding properties.J Bacteriol. 1996; 178: 6632-6634Crossref PubMed Google Scholar]. The strong conservation of this loop in the STAS domains (Figure 1) suggests that this domain could possess general NTP-binding activity. The conserved loop is probably involved in phosphate-binding and the β-sheet scaffold could accommodate the rest of the NTP molecule. This mode of ligand binding resembles lipid binding by Sec14 domains, which have the same structural fold as SPOIIAA but show no detectable sequence similarity to the STAS domain [17Aravind L. Neuwald A.F. Ponting C.P. Sec14p-like domains in NF1 and Dbl-like proteins indicate lipid regulation of Ras and Rho signaling.Curr Biol. 1999; 9: R195-R197Abstract Full Text Full Text PDF PubMed Google Scholar, 18Hubbard T.J. Ailey B. Brenner S.E. Murzin A.G. Chothia C. SCOP: a structural classification of proteins database.Nucleic Acids Res. 1999; 27: 254-256Crossref PubMed Scopus (186) Google Scholar]. Notably, one of the mutations in PDS has been mapped to the predicted phosphate-binding loop and probably results in its disruption (Figure 1, Figure 2). The presence of a predicted NTP-binding domain in the cytoplasmic portions of anion transporters indicates that anion transport could be regulated by intracellular concentrations of GTP and/or ATP. The NTPs are likely to elicit specific conformational changes in the STAS domain through binding and/or hydrolysis. The critical role of the STAS domain in anion transporters is supported by a number of mutations in PDS and DTD that map to this domain [3Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. et al.Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).Nat Genet. 1997; 17: 411-422Crossref PubMed Scopus (948) Google Scholar, 19Hastbacka J. Superti-Furga A. Wilcox W.R. Rimoin D.L. Cohn D.H. Lander E.S. Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias.Am J Hum Genet. 1996; 58: 255-262PubMed Google Scholar, 20Superti-Furga A. Rossi A. Steinmann B. Gitzelmann R. A chondrodysplasia family produced by mutations in the diastrophic dysplasia sulfate transporter gene: genotype/phenotype correlations.Am J Med Genet. 1996; 63: 144-147Crossref PubMed Scopus (78) Google Scholar]. Experimental testing of these predictions, for which bacterial transporters with the same domain architecture could serve as a model, should clarify the regulation of these important transporters, which appears to be more complex than previously suspected. We detected ASA-like STAS domain proteins in some bacteria (for example Escherichia coli) that lack the typical sigma regulatory system and new members in Gram-positive bacteria that are fused to another ligand-binding domain — the PAS domain (Figure 1). These STAS proteins could represent new bacterial regulatory systems. We thank Michael Yudkin for helpful discussions.