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Substrate-induced changes in the structural properties of LacY.

Abstract：

:The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H(+) across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H(+)-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H(+)- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H(+) symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154→Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154→Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Serdiuk T,Madej MG,Sugihara J,Kawamura S,Mari SA,Kaback HR,Müller DJ

doi

10.1073/pnas.1404446111

subject

Has Abstract

pub_date

2014-04-22 00:00:00

pages

E1571-80

issue

eissn

0027-8424

issn

1091-6490

pii

1404446111

journal_volume

111

pub_type

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Substrate-induced changes in the structural properties of LacY.