Solution Structure of the Guanine Nucleotide-binding STAS Domain of SLC26-related SulP Protein Rv1739c from Mycobacterium tuberculosis

Alan C. Rigby,Liwen Ye,Tom Alber,Christina E. Baer,Kumaran Shanmugasundaram,Seth L. Alper,Alok K. Sharma

doi:10.1074/jbc.m110.165449

Abstract

The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded β-sheet with five interspersed α-helices, resembling the anti-σ factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-σ factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-σ factor antagonists, may not mediate signals via phosphorylation.

Highlights

The majority of bacterial SulP proteins and all known eukaryotic SulP/SLC26 proteins carry a C-terminal cytoplasmic “sulfate transporter anti-␴ factor antagonist” (STAS)4 domain that shares distant sequence homology with bacterial anti-␴ factor antagonist proteins [19, 20]
Using intrinsic fluorescence quench data and 1H-15N chemical shift perturbation (CSP) experiments, we demonstrate that the monomeric STAS domain binds guanine nucleotides at physiological concentrations
Xaa-Pro peptide bond conformations were predicted from chemical shift values of 15N, 1H⌵, 1H␣, 13C␣, 13C␤, and 13CO resonances using PROMEGA revealed a transconfiguration for each of the 5 Pro residues of Rv1739c STAS

Summary

EXPERIMENTAL PROCEDURES

Purification of Rv1739c STAS Domain—A polypeptide encompassing aa 437–560 of Rv1739c was overexpressed for study of the STAS domain, based on alignment with STAS domains from E. coli ychM and Sultr1.2, and with structurally characterized STAS domains from anti-␴ factor antagonists of B. subtilis, Bacillus sphericus, and T. maritima 1442. Steady-state intrinsic fluorescence intensity of RV1739c STAS was recorded at ␭em 290 – 400 nm at 2-nm intervals with fixed ␭ex ϭ 280 nm (SpectraMax M5, Molecular Devices, Sunnyvale, CA) in the presence of sequentially increasing nucleotide concentrations or titrated with buffer A alone. Rv1739c STAS fluorescence at each nucleotide concentration was corrected for dilution and for inner filter effect contributions of added nucleotide, as described [45]. The effects of identical sequential increases in added nucleotide were assessed on the intrinsic fluorescence of a mixture of free tryptophan plus 3 molar eq of free tyrosine (the 1:3 Trp:Tyr molar ratio reflected in the native Rv1739c STAS amino acid sequence), with identical data acquisition parameters and instrument settings. Total Intraresidue (͉i-jϭ 0) Sequential (͉i-jϭ 1) Medium range (1 Ͻ (͉i-j) Յ 4) Long range (͉i-j) Ͼ 4)

All heavy atoms

RESULTS

DISCUSSION