Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli

Aileen L. Green, Heather A. Hrodey, Robert J. Brooker

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9 Scopus citations

Abstract

Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 8 (TMS-8) play a critical role in conformational changes associated with lactose transport (Green, A. L., and Brooker, R. J. [2001] Biochemistry 40, 12220-12229). Substitutions at positions 261, 265, 268, 272, and 276, which form a continuous stripe along TMS-8, were markedly defective for lactose transport velocity. In the current study, three single mutants (F261D, N272Y, N272L) and a double mutant (T265Y/276Y) were chosen as parental strains for the isolation of mutants that restored transport function. A total of 68 independent mutants were isolated and sequenced. Forty-four were first-site revertants in which the original mutation was changed back to the wild-type residue or to a residue with a similar side-chain volume. The other 24 mutations were second-site suppressors in TMS-2 (Q60L, Q60P), loop 2/(L70H), TMS-7 (V229G/), TMS-8 (F261L), and TMS-11 (F354V, C355G). On the basis of their locations, the majority of the second-site suppressors can be interpreted as improving the putative TMS-2/MS-7/MS-11 interface to compensate for conformational defects imposed by mutations in TMS-8 that disrupt the putative TMS-1/MS-5/MS-8 interface. Overall, this paper suggests that the TMS-2/MS-7/MS-11 interface is more important from a functional point of view, even though there is compelling evidence for structural symmetry between the two halves of the permease.

Original languageEnglish (US)
Pages (from-to)11226-11233
Number of pages8
JournalBiochemistry
Volume42
Issue number38
DOIs
StatePublished - Sep 30 2003

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