Transmembrane remote conformational suppression of the Gly-332 mutation of the Tn10-encoded metal-tetracycline/H+ antiporter.

Gly-332 is a conformationally important residue of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)), which was found by random mutagenesis and confirmed by site-directed mutagenesis. A bulky side chain at position 332 is deleterious to the transport function. A spontaneous second-site suppressor revertant was isolated from G332S mutant and identified as the Ala-354-->Asp mutant. Gly-332 and Ala-354 are located on opposite ends of transmembrane segment XI. As judged from [14C]NEM binding to Cys mutants, the residue at position 354, which is originally exposed to water, was buried in the membrane by a G332S mutation through a remote conformational change of transmembrane segment XI. This effect is the same as that of a G62L mutation at position 30 through transmembrane segment II [Kimura, T., Sawai, T. and Yamaguchi, A. (1997) Biochemistry 36, 6941-6946]. Interestingly, the G332S mutation was also suppressed by the L30S mutation, and the G62L mutation was moderately suppressed by the A354D mutation. These results indicate the presence of a close conformational relationship between the flanking regions of the transmembrane segments II and XI.