The long-range goal of this proposal is to obtain a crystal structure(s) of a new conformation of the lactose permease of Escherichia coli (LacY) in order to understand the mechanism of lactose/H+ symport. LacY is a paradigm for a huge group of structurally related membrane transport proteins (the Major Facilitator Superfamily), as well as for membrane proteins in general. Our first X-ray crystal structure of a conformationally crippled mutant of LacY (C154G) represents a major breakthrough as the first structure of a cation-coupled symporter. In the past grant period, we also solved an X-ray structure of wild-type LacY to a resolution of 3.6 ?, which represents another highly significant breakthrough, as the accomplishment took well over a decade and required development of a new approach that has general applicability. In addition, we improved the resolution of the C154G LacY structure to a resolution of 2.95 ?. However, all available structures from the wild type and the conformationally crippled mutant display the same global fold. All structures are composed of symmetrical N- and C-terminal domains, each with 6 transmembrane α-helices most of which are irregular. There is a large internal hydrophilic cavity open to the cytoplasmic side, which clearly represents an inward-facing conformation, as the periplasmic side is tightly closed. The residues that play major roles in galactopyranoside recognition and H+ translocation are clustered near the apex of the cavity and are inaccessible from the periplasmic side. A mechanism consistent with the structure and various biochemical and biophysical approaches has been proposed, the heart of which is alternative accessibility of the sugar-binding site to either side of the membrane. Therefore, it is critical to obtain structures in a different conformations. We have obtained diffracting crystals of mutants and constructs that are approaching a resolution suitable for structure determination. The main aims of the proposal are (i) to obtain structures of other conformations of LacY;(ii) to obtain a structure of LacY that diffracts to a resolution sufficient to visualize bound water. We will combine mutagenesis and chemical modification to induce conformations different from the inward-facing confirmation. The proposed structures will be invaluable for understanding the mechanism of cation-coupled membrane transporters, a class of proteins that plays essential roles in many cellular functions and has broad impact on biology and medicine.
Membrane proteins represent a very significant percentage of the genomes sequenced, and although they are involved in a multitude of essential cellular functions and are targets for the world?s most widely prescribed drugs, their structures are grossly underrepresented. The lactose permease (LacY), which physiologically catalyzes the coupled translocation of lactose and a hydrogen atom across the membrane of the bacterium Escherichia coli, represents a well-known model system for a huge family of related membrane transport proteins, many of which are clinically important. LacY has been used to develop numerous techniques for studying of this type of membrane transport protein. In order to under its mechanism of action, it is essential to obtain structures of LacY in more than the single form that we have revealed, which is the purpose of this proposal.
| Serdiuk, Tetiana; Balasubramaniam, Dhandayuthapani; Sugihara, Junichi et al. (2016) YidC assists the stepwise and stochastic folding of membrane proteins. Nat Chem Biol 12:911-917 |
| Madej, M Gregor (2015) Comparative Sequence-Function Analysis of the Major Facilitator Superfamily: The ""Mix-and-Match"" Method. Methods Enzymol 557:521-49 |
| Kumar, Hemant; Finer-Moore, Janet S; Kaback, H Ronald et al. (2015) Structure of LacY with an ?-substituted galactoside: Connecting the binding site to the protonation site. Proc Natl Acad Sci U S A 112:9004-9 |
| Serdiuk, Tetiana; Sugihara, Junichi; Mari, Stefania A et al. (2015) Observing a lipid-dependent alteration in single lactose permeases. Structure 23:754-61 |
| Kaback, H Ronald (2015) A chemiosmotic mechanism of symport. Proc Natl Acad Sci U S A 112:1259-64 |
| Smirnova, Irina; Kasho, Vladimir; Kaback, H Ronald (2014) Real-time conformational changes in LacY. Proc Natl Acad Sci U S A 111:8440-5 |
| Madej, M Gregor; Sun, Linfeng; Yan, Nieng et al. (2014) Functional architecture of MFS D-glucose transporters. Proc Natl Acad Sci U S A 111:E719-27 |
| Serdiuk, Tetiana; Madej, M Gregor; Sugihara, Junichi et al. (2014) Substrate-induced changes in the structural properties of LacY. Proc Natl Acad Sci U S A 111:E1571-80 |
| Smirnova, Irina; Kasho, Vladimir; Jiang, Xiaoxu et al. (2014) Outward-facing conformers of LacY stabilized by nanobodies. Proc Natl Acad Sci U S A 111:18548-53 |
| Jiang, Xiaoxu; Villafuerte, Maria Katerina R; Andersson, Magnus et al. (2014) Galactoside-binding site in LacY. Biochemistry 53:1536-43 |
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