| Research Abstract |
Bacterial metal-tetracycline/H^+ antiporter (Tet (B)) is one of typical xenobiotic exporters which have been widely found in living organisms as basic devices for host defense mechanism. In this study, we investigated the molecular structure of Tet (B) and the pathway of the drug extrusion. Cysteine-scanning mutants of Tet (B) were constructed on the basis of the Cys-free mutant. When the Cys residue is located on the outside of the membrane, the binding of membrane-permeable SH reagent, [14C]NEM,to the Cys residue of Tet (B) expressed in the intact cells should be competitively inhibited by an membrane-impermeable reagent, AMS.On the contrary, when it is located inside, the binding should not be inhibited. By means of this method, we experimentaly proved the 12 membrane-spanning structure of Tet (B). Then, we succeeded to determine the exact boundary between the membrane-spanning segments and the water-extruding loops by means of the reactivity of [14C] NEM with the Cys-scanning mutants. The Cys residues located in the membrane-spnanning reagion showed no or very low reactivity with NEM,however, the Cys residues on the water-exposed loops showed high reactivity. The confomational change during the substrate translocation or by the mutation was also detected by means of the reactivity of [14C] NEM with Cys residues : (1) Tet (B) underwent the substrate-induced conformational change from the inside-closed/outside-open conformation to the inside-open/outside-closed one, and (2) the remote-conformational change caused by a mutation was suppressed by a second-site suppressor mutation. On the basis of these protein-engineering studies, we proposed the 3D model of Tet (B) protein.
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