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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Sechi, Salvatore
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University of California Los Angeles
Schools of Medicine
Los Angeles
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Grytsyk, Natalia; Sugihara, Junichi; Kaback, H Ronald et al. (2017) pKa of Glu325 in LacY. Proc Natl Acad Sci U S A 114:1530-1535
Smirnova, Irina; Kasho, Vladimir; Jiang, Xiaoxu et al. (2017) An Asymmetric Conformational Change in LacY. Biochemistry 56:1943-1950
Jiang, Xiaoxu; Andersson, Magnus; Chau, Bryan T et al. (2016) Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY. Biochemistry 55:4326-32
Hariharan, Parameswaran; Andersson, Magnus; Jiang, Xiaoxu et al. (2016) Thermodynamics of Nanobody Binding to Lactose Permease. Biochemistry 55:5917-5926
Madej, M Gregor (2015) Comparative Sequence-Function Analysis of the Major Facilitator Superfamily: The ""Mix-and-Match"" Method. Methods Enzymol 557:521-49
Liebeskind, Benjamin J; Hillis, David M; Zakon, Harold H (2015) Convergence of ion channel genome content in early animal evolution. Proc Natl Acad Sci U S A 112:E846-51
Smirnova, Irina; Kasho, Vladimir; Jiang, Xiaoxu et al. (2015) Transient conformers of LacY are trapped by nanobodies. Proc Natl Acad Sci U S A 112:13839-44
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

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