Role of protons in sugar binding to LacY. Proc Natl Acad Sci USA

Departments of Physiology and Microbiology, Immunology and Molecular Genetics and Molecular Biology Institute, University of California, Los Angeles, CA 90095.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2012; 109(42):16835-40. DOI: 10.1073/pnas.1214890109
Source: PubMed

ABSTRACT WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H(+) translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H(+) translocation is not accompanied by release of an H(+) on the interior of the proteoliposomes. Because the pK(a) value for galactoside binding is ∼10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5-8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H(+) transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (k(off)) and the forward rate (k(on)) are slower in D(2)O than in H(2)O (KIE is ∼2), and, as a result, no change in affinity (K(d)) is observed. Alkaline conditions enhance the effect of D(2)O on k(off), the KIE increases to 3.6-4.0, and affinity for sugar increases compared with H(2)O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e.g., Glu325 → Gln) exhibit the same KIE (1.5-1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between k(off) and D(2)O concentration at neutral and alkaline pH, indicating that internal transfer of a single H(+) is involved in the KIE.

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Available from: Junichi Sugihara, Apr 02, 2015
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    • "j.str.2014.06.008 support the notion that acidic residues in this cluster are important for protonation in response to substrate binding rather than for the substrate binding per se. A similar cluster of polar residues has also been observed in the galactoside/H + symporter LacY, where substrate binding and protonation are coupled in an obligatory manner (Smirnova et al., 2012; Zhou et al., 2012). "
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    ABSTRACT: E. coli YbgH belongs to the family of proton-dependent oligopeptide transporters (POTs), a subfamily of the major facilitator superfamily (MFS) of secondary active transporters. Like other MFS transporters, POT proteins switch between two major conformations during substrate transport. Apart from possessing a canonical 12-helix, two-domain transmembrane (TM) core, prokaryotic POT proteins usually have two TM helices inserted between the two domains. Here we determined the crystal structure of YbgH in its inward-facing conformation. Our structure-based functional studies investigated the roles of both the POT signature motif 2 and the inserted interdomain TM helix pair in the stabilization and regulation of the major conformational change in MFS/POT transporters. Furthermore, of all the proton-titratable amino acid residues, Glu21 is the only conserved one (among POTs) located in the central cavity and is critical for in vivo transport. Together, our results support the notion that MFS symporters utilize a transport mechanism based on substrate-protonation coupling.
    Structure 07/2014; 22(8). DOI:10.1016/j.str.2014.06.008 · 6.79 Impact Factor
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    • "7. A mechanism for proton coupled transport within the PTR family A large body of experimental data collected on the lactose permease, LacY from E. coli, has led to a clearer picture emerging for the role of protons in the sugar:proton symporters [66]. During uphill transport, or the protein working under physiological conditions of lactose accumulation , protonation is required for LacY to bind lactose, whereupon it is the energy of sugar binding and dissociation into the interior of the cell that is predicted to drive the conformational changes that result in transport [67] [68]. "
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    Biochimica et Biophysica Acta (BBA) - General Subjects 01/2014; · 3.83 Impact Factor
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    ABSTRACT: The major facilitator superfamily (MFS) is one of the largest groups of secondary active transporters conserved from bacteria to humans. MFS proteins selectively transport a wide spectrum of substrates across biomembranes and play a pivotal role in multiple physiological processes. Despite intense investigation, only seven MFS proteins from six subfamilies have been structurally elucidated. These structures were captured in distinct states during a transport cycle involving alternating access to binding sites from either side of the membrane. This review discusses recent progress in MFS structure analysis and focuses on the molecular basis for substrate binding, co-transport coupling, and alternating access.
    Trends in Biochemical Sciences 03/2013; 38(3):151-9. DOI:10.1016/j.tibs.2013.01.003 · 13.52 Impact Factor
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