Real-time Conformational Changes in LacY

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2014 May 30

PMID 24872451

Citations 16

Authors

Irina Smirnova

Vladimir Kasho

H Ronald Kaback

Affiliations

Soon will be listed here.

Abstract

Galactoside/H(+) symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein. Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s(-1), independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity.

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References

Smirnova I, Kasho V, Sugihara J, Kaback H . Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation. Proc Natl Acad Sci U S A. 2013; 110(22):8876-81. PMC: 3670396. DOI: 10.1073/pnas.1306849110. View

Janz J, Farrens D . Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus. J Biol Chem. 2004; 279(28):29767-73. DOI: 10.1074/jbc.M402567200. View

Guan L, Mirza O, Verner G, Iwata S, Kaback H . Structural determination of wild-type lactose permease. Proc Natl Acad Sci U S A. 2007; 104(39):15294-8. PMC: 2000551. DOI: 10.1073/pnas.0707688104. View

Nie Y, Ermolova N, Kaback H . Site-directed alkylation of LacY: effect of the proton electrochemical gradient. J Mol Biol. 2007; 374(2):356-64. PMC: 2224895. DOI: 10.1016/j.jmb.2007.09.006. View

Smirnova I, Kasho V, Kaback H . Lactose permease and the alternating access mechanism. Biochemistry. 2011; 50(45):9684-93. PMC: 3210931. DOI: 10.1021/bi2014294. View

Sun J, Wu J, Carrasco N, Kaback H . Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli. Biochemistry. 1996; 35(3):990-8. DOI: 10.1021/bi952166w. View

Mirza O, Guan L, Verner G, Iwata S, Kaback H . Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY. EMBO J. 2006; 25(6):1177-83. PMC: 1422171. DOI: 10.1038/sj.emboj.7601028. View

Nie Y, Kaback H . Sugar binding induces the same global conformational change in purified LacY as in the native bacterial membrane. Proc Natl Acad Sci U S A. 2010; 107(21):9903-8. PMC: 2906848. DOI: 10.1073/pnas.1004515107. View

Majumdar D, Smirnova I, Kasho V, Nir E, Kong X, Weiss S . Single-molecule FRET reveals sugar-induced conformational dynamics in LacY. Proc Natl Acad Sci U S A. 2007; 104(31):12640-5. PMC: 1937519. DOI: 10.1073/pnas.0700969104. View

10.

Smirnova I, Kasho V, Sugihara J, Kaback H . Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding. Proc Natl Acad Sci U S A. 2011; 108(37):15147-51. PMC: 3174601. DOI: 10.1073/pnas.1112157108. View

11.

Kaback H, Dunten R, Frillingos S, Venkatesan P, Kwaw I, Zhang W . Site-directed alkylation and the alternating access model for LacY. Proc Natl Acad Sci U S A. 2006; 104(2):491-4. PMC: 1766412. DOI: 10.1073/pnas.0609968104. View

12.

Yao X, Parnot C, Deupi X, Ratnala V, Swaminath G, Farrens D . Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor. Nat Chem Biol. 2006; 2(8):417-22. DOI: 10.1038/nchembio801. View

13.

Islas L, Zagotta W . Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence. J Gen Physiol. 2006; 128(3):337-46. PMC: 2151569. DOI: 10.1085/jgp.200609556. View

14.

Mansoor S, Farrens D . High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane. Biochemistry. 2004; 43(29):9426-38. DOI: 10.1021/bi036259m. View

15.

Smirnova I, Kasho V, Choe J, Altenbach C, Hubbell W, Kaback H . Sugar binding induces an outward facing conformation of LacY. Proc Natl Acad Sci U S A. 2007; 104(42):16504-9. PMC: 2034228. DOI: 10.1073/pnas.0708258104. View

16.

Herzlinger D, Viitanen P, Carrasco N, Kaback H . Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein. Biochemistry. 1984; 23(16):3688-93. DOI: 10.1021/bi00311a018. View

17.

Abramson J, Smirnova I, Kasho V, Verner G, Kaback H, Iwata S . Structure and mechanism of the lactose permease of Escherichia coli. Science. 2003; 301(5633):610-5. DOI: 10.1126/science.1088196. View

18.

Madej M, Soro S, Kaback H . Apo-intermediate in the transport cycle of lactose permease (LacY). Proc Natl Acad Sci U S A. 2012; 109(44):E2970-8. PMC: 3497813. DOI: 10.1073/pnas.1211183109. View

19.

Smirnova I, Kasho V, Kaback H . Direct sugar binding to LacY measured by resonance energy transfer. Biochemistry. 2006; 45(51):15279-87. PMC: 2566955. DOI: 10.1021/bi061632m. View

20.

Tsukamoto H, Farrens D, Koyanagi M, Terakita A . The magnitude of the light-induced conformational change in different rhodopsins correlates with their ability to activate G proteins. J Biol Chem. 2009; 284(31):20676-83. PMC: 2742832. DOI: 10.1074/jbc.M109.016212. View