Structural determination of wild-type lactose permease.
ABSTRACT Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H(+) binding. To initiate transport, binding of sugar and/or an H(+) electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
SourceAvailable from: Ajay Nooka[Show abstract] [Hide abstract]
ABSTRACT: Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints (1). Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues (2), GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells (3). Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma (3). GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia (CLL) and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4 (4,5). Our current study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported (6). While this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of eighteen million compounds. Despite 68% homology (7) between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4 selective inhibitors as anti-cancer therapeutics. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 04/2015; DOI:10.1074/jbc.M114.628826 · 4.60 Impact Factor
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ABSTRACT: The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. The paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and the most recent structure of an almost occluded conformation confirm many conclusions from previous studies. One fundamentally important problem for understanding the mechanism of secondary active transport is the identification and physical localization of residues involved in substrate and H(+) binding. This information is exceptionally difficult to obtain with the MFS because of the broad sequence diversity among the members. The increasing number of solved MFS structures has led to the recognition of a common feature: inverted structure-repeat, formed by fused triple-helix domains with opposite orientation in the membrane. The presented method here exploits this feature to predict functionally homologous positions of known relevant positions in LacY. The triple-helix motifs are aligned in combinatorial fashion so as to detect substrate and H(+)-binding sites in symporters that transport substrates, ranging from simple ions like phosphate to more complex disaccharides. © 2015 Elsevier Inc. All rights reserved.Methods in Enzymology 03/2015; 557. DOI:10.1016/bs.mie.2014.12.015 · 2.19 Impact Factor
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ABSTRACT: The Na(+)/multivitamin transporter (SMVT) is a member of the solute:sodium symporter family that catalyzes the Na(+)-dependent uptake of the structurally diverse water-soluble vitamins pantothenic acid (vitamin B5) and biotin (vitamin H), α-lipoic acid-a vitamin-like substance with strong antioxidant properties-and iodide. The organic substrates of SMVT play central roles in the cellular metabolism and are, therefore, essential for normal human health and development. For example, biotin deficiency leads to growth retardation, dermatological disorders, and neurological disorders. Animal studies have shown that biotin deficiency during pregnancy is directly correlated to embryonic growth retardation, congenital malformation, and death of the embryo. This chapter focuses on the structural and functional features of the human isoform of SMVT (hSMVT); the discovery of which was greatly facilitated by the cloning and expression of hSMVT in tractable expression systems. Special emphasis will be given to mechanistic implications of the transport process of hSMVT that will inform our understanding of the molecular determinants of hSMVT-mediated transport in dynamic context to alleviate the development and optimization of hSMVT as a multipotent platform for drug delivery. © 2015 Elsevier Inc. All rights reserved.Vitamins & Hormones 01/2015; 98:63-100. DOI:10.1016/bs.vh.2014.12.003 · 1.78 Impact Factor