An Early Event in the Transport Mechanism of LacY Protein: Interaction Between Helices V and I

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2011 Jul 7

PMID 21730060

Citations 10

Authors

Yonggang Zhou

M Gregor Madej

Lan Guan

Yiling Nie

H Ronald Kaback

Affiliations

Soon will be listed here.

Abstract

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg(144) and Trp(151), two residues that play direct roles in sugar recognition and binding, as well as Cys(154), which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro(28) → Cys/Cys(154), Pro(28) → Cys/Val(158) → Cys, and Phe(29) → Cys/Val(158) → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)(2)-1), 3-4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)(2)-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.

Citing Articles

Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY.

Jiang X, Andersson M, Chau B, Wong L, Villafuerte M, Kaback H Biochemistry. 2016; 55(31):4326-32.

PMID: 27438891 PMC: 5456280. DOI: 10.1021/acs.biochem.6b00666.

Substrate-induced changes in the structural properties of LacY.

Serdiuk T, Madej M, Sugihara J, Kawamura S, Mari S, Kaback H Proc Natl Acad Sci U S A. 2014; 111(16):E1571-80.

PMID: 24711390 PMC: 4000801. DOI: 10.1073/pnas.1404446111.

Flexible gates generate occluded intermediates in the transport cycle of LacY.

Stelzl L, Fowler P, Sansom M, Beckstein O J Mol Biol. 2014; 426(3):735-51.

PMID: 24513108 PMC: 3905165. DOI: 10.1016/j.jmb.2013.10.024.

Protonation drives the conformational switch in the multidrug transporter LmrP.

Masureel M, Martens C, Stein R, Mishra S, Ruysschaert J, Mchaourab H Nat Chem Biol. 2013; 10(2):149-55.

PMID: 24316739 PMC: 4749020. DOI: 10.1038/nchembio.1408.

Evolutionary mix-and-match with MFS transporters II.

Madej M, Kaback H Proc Natl Acad Sci U S A. 2013; 110(50):E4831-8.

PMID: 24259711 PMC: 3864288. DOI: 10.1073/pnas.1319754110.

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