Abstract
Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.
Highlights
The Tn10-encoded metal-tetracycline/Hϩ antiporter (TetA(B))1 is a typical bacterial drug export protein [2, 3], and its molecular mechanism and molecular structure have been studied as a paradigm of antiporter-type drug exporters including bacterial multidrug exporters (4 – 6)
In our previous paper [1], we reported that both G62L, which is in cytoplasmic loop 2–3, and G332S, which is in cytoplasmic loop 10 –11, are suppressed by the second-site mutations of both L30S and A354D, which are located in periplasmic loop 1–2 and loop 11–12, respectively
We could detect intramolecular cross-linking as an SDS-PAGE mobility shift of the double mutant after oxidation; we introduced a Cys residue into each of the six periplasmic loop regions and constructed double Cys mutants
Summary
TetA(B), Tn10-encoded metal-tetracycline/Hϩ antiporter; PAGE, polyacrylamide gel electrophoresis; PDM, phenylenedimaleimide; BMH, 1,6-bis(maleimido)hexane; MFS, major facilitator superfamily; CuPh, Cu2ϩ/o-phenanthroline; NEM, N-ethylmaleimide. Second-site mutations at Leu and Ala354 prevent the remote conformational distortion caused by the G62L and G332S mutations [1, 16] These results indicate the possibility that positions 30 and 354 are spacially close to each other. We could detect intramolecular cross-linking as an SDS-PAGE mobility shift of the double mutant after oxidation; we introduced a Cys residue into each of the six periplasmic loop regions and constructed double Cys mutants. The results of the cross-linking assay indicate that the two ends, P1 and P6, are close to each other and that one of the central loops, P4, is close to P1 Such a periplasmic loop arrangement is similar to that of lac permease [17], indicating that the MFS transporters may have a general three-dimensional structure despite the difference in the proton-coupling direction
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