We aim to understand the dynamics of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily that contains for example the vesicular monoamine transporter (VMAT), as well as GLUT1, which transports glucose across the blood brain barrier. Like channels and ABC transporters, ion gradient-coupled membrane transport proteins are also highly relevant to human physiology and disease (e.g. depression, epilepsy, diabetes, multidrug resistance). Also of note, at least two of the most widely prescribed drugs in the world [serotonin selective reuptake inhibitors (SSRIs) and gastric proton pump inhibitors (PPIs)], are targeted to membrane transport proteins. Near-atomic level structures of wild-type LacY, as well as a conformationally restricted mutant, and a library of single-Cys mutants and many other mutants, have provided critical information regarding the structure and mechanism of LacY. The protein consists of two pseudo-symmetrical bundles of 6 transmembrane helices, mostly irregularly shaped, surrounding a large, hydrophilic internal cavity open to the cytoplasmic side only. The periplasmic side is tightly packed so that the sugar- and H+-binding sites are inaccessible from this side. The structure leads a priori to the notion that the mechanism involves a global conformational change in which the inward- facing cavity closes with opening of a periplasmic pathway so that the binding sites become alternatively accessible from either side of the membrane (i.e., the alternating access model). Although the structures reveal a number of novel observations and confirm many findings, we are just beginning to gain insight into the dynamics of LacY with respect to conformational states and their transitions during ligand binding and turnover. In the future, we will address fundamental questions such as rates of opening and closing of the cavities and the effect of reconstitution and the H+ electrochemical gradient by applying structure-based techniques developed in this laboratory and now used internationally. Integration of the findings with data currently available will facilitate far greater insight into the mechanism of galactoside/H+ symport and have an even greater influence on the important field of membrane transport.

Public Health Relevance

Membrane proteins represent a highly significant percentage of the genomes sequenced, they are highly relevant to human physiology and disease (e.g. depression, epilepsy, diabetes, multidrug resistance), and they are major drug targets [e.g. selective serotonin reuptake inhibitors (SSRIs)], but our understanding of their molecular mechanisms lags far behind that of soluble proteins. The lactose permease (LacY), a well-known membrane transport protein, is a model for a family of >10,000 related transport proteins (the Major Facilitator Superfamily) many of which are clinically important (e.g. VMAT, the GLUTs). The advances this laboratory has achieved represent a major breakthrough in our understanding of the general principles of membrane transport, and we are now beginning to gain insight into dynamics with respect to alternating accessibility of binding sites to either side of the membrane, rates of important conformational changes and their transitions during sugar/H+ symport.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK051131-19
Application #
8663228
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Sechi, Salvatore
Project Start
1996-06-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
19
Fiscal Year
2014
Total Cost
$404,071
Indirect Cost
$141,687
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Kumar, Hemant; Kasho, Vladimir; Smirnova, Irina et al. (2014) Structure of sugar-bound LacY. Proc Natl Acad Sci U S A 111:1784-8
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
Ethayathulla, Abdul S; Yousef, Mohammad S; Amin, Anowarul et al. (2014) Structure-based mechanism for Na(+)/melibiose symport by MelB. Nat Commun 5:3009
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
Jiang, Xiaoxu; Villafuerte, Maria Katerina R; Andersson, Magnus et al. (2014) Galactoside-binding site in LacY. Biochemistry 53:1536-43
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; Dang, Shangyu; Yan, Nieng et al. (2013) Evolutionary mix-and-match with MFS transporters. Proc Natl Acad Sci U S A 110:5870-4
Jiang, Xiaoxu; Driessen, Arnold J M; Feringa, Ben L et al. (2013) The periplasmic cavity of LacY mutant Cys154ýýýGly: how open is open? Biochemistry 52:6568-74
Zhu, Lu; Kaback, H Ronald; Dalbey, Ross E (2013) YidC protein, a molecular chaperone for LacY protein folding via the SecYEG protein machinery. J Biol Chem 288:28180-94
Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi et al. (2013) Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation. Proc Natl Acad Sci U S A 110:8876-81

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