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Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The structural resemblance and functional difference in the role of the duplicated sequence motif between hydrophobic segments 2 and 3 and segments 8 and 9.

Akihito Yamaguchi,Tomomi Kimura,Y. Someya,T. Sawai

Published 1993 in Journal of Biological Chemistry

ABSTRACT

The properties of site-directed mutants as to the putative hydrophilic loop region between hydrophobic segments 2 and 3 in the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TET) were reported in our previous paper (Yamaguchi, A., Someya, Y., and Sawai, T. (1992) J. Biol. Chem. 267, 19155-19162). The loop between hydrophobic segments 8 and 9 contains a conserved sequence motif, GXXXXKXGEK, which is a derivative of the sequence motif, GXXXXRXGRR, in loop2-3. Site-directed mutagenesis studies on loop8-9 revealed that the two loops exhibit significant structural resemblance, that is, 1) when the Gly residue at the eighth position in each loop was replaced by various amino acid residues, the residual activity of the resultant mutants corresponded well to the beta-turn propensity of the substituent, 2) the Cys mutant as to the fourth position in each loop was most profoundly inactivated by N-ethylmaleimide among 10 Cys mutants as to each loop, and 3) the reactivity of a Cys residue introduced at the third position in loop8-9 with N-ethylmaleimide was lower than that in the case of the other Cys mutants, probably due to the residue being partially cryptic as to the attack of the reagent, similar to in the case of the corresponding residue in loop2-3, the latter being entirely cryptic. The Gly at the first position in loop8-9 is less important than the corresponding Gly in loop2-3, however, since the TET protein suffered a loss of activity when a bulky side chain was introduced at the first position in loop8-9 as well as in loop2-3, the structural roles of the 2 glycines are likely to be similar. These findings suggested that loop2-3 and loop8-9 may occupy similar positions in the three-dimensional structure of the TET protein. On the other hand, the two loops showed a significant functional difference; the negative charge of Asp66 and the positive charge of Arg70 in loop2-3 were essential for the transport function, but, in contrast, there was no functionally essential residue in loop8-9, indicating that loop2-3 may form an "active" leaflet in the TET protein, while loop8-9 may be a "silent" counterpart.

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