title: Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA.
abstract: We have previously shown that the C-terminal cystathionine beta-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I(in)). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5-7 degrees C and the temperature coefficient Q(10) was lowered from 8 to approximately 1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, and Zernike Institute for Advanced Materials & Centre for Synthetic Biology, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Nik A B N
authors.initials: NA
authors.lastname: Mahmood
authors.forename: Esther
authors.initials: E
authors.lastname: Biemans-Oldehinkel
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman