The aim of this proposal is to develop an atomic-level understanding of the mechanism of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), an important model for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. The MFS contains many members that have clinical significance such as the vesicular monoamine transporter, as well as the GLUTs, which facilitate glucose transport into many different cells in the body. Like channels and ABC transporters, symporters are also highly relevant to human health (e.g. transport across epithelia, synaptic function) and disease (e.g. depression, epilepsy, diabetes, multidrug resistance). Also notable, at least two of the most widely used pharmaceuticals in the world [serotonin selective reuptake inhibitors (SSRIs) and gastric proton pump inhibitors (PPIs)] are targeted to membrane transport proteins. Also important, the innovative methods developed by the PI to study LacY have been applied directly to such important human membrane proteins as GLUT1 and G-Protein-Coupled Receptors (GPCRs). However, despite a number of structures of MFS members, including 7 of LacY, the mechanism of this dynamic protein is not completely understood. It has been demonstrated that sugar binding to highly dynamic protonated LacY triggers a global conformational change in which sugar- and H+-binding sites gain alternating access to either side of the membrane. Sugar binding and dissociation drive this conformational change through an induced-fit mechanism, while the proton electrochemical gradient enhances the rate of deprotonation. Therefore, LacY behaves much like an enzyme except that the transition state(s) involves the protein rather than the substrate. X-ray structures of LacY inward- and almost occluded outward-facing conformations provide the structural basis for studying the alternating access mechanism. The alternating access mechanism has been documented unequivocally by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches pioneered in this laboratory, and by using kinetic data obtained in real time for several steps in the transport cycle. Thirty-one Camelid nanobodies now in our possession allow stabilization of LacY in different intermediate states that will provide an in-depth understanding of structural changes underlying the symport mechanism. It should be emphasized that the approaches and the methodologies described here for LacY are already being applied to other membrane transport proteins and can be applied to membrane proteins in general.

Public Health Relevance

Membrane proteins represent ~25-30% of the genomes sequenced, they are highly relevant to human physiology (e.g. transport across epithelia, synaptic function) and disease (e.g. depression, epilepsy, diabetes, multidrug resistance), and they are targeted by at least two of the most widely prescribed drugs in the world (serotonin selective reuptake inhibitors and gastric proton pump inhibitors). The tools developed by this laboratory will yield an in-depth understanding of the principles behind H+-coupled membrane transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM120043-01S1
Application #
9355287
Study Section
Special Emphasis Panel (ZRG1-MDCN-C (05)S)
Program Officer
Chin, Jean
Project Start
2016-09-01
Project End
2020-05-31
Budget Start
2016-09-01
Budget End
2017-05-31
Support Year
1
Fiscal Year
2016
Total Cost
$95,308
Indirect Cost
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
Smirnova, Irina; Kasho, Vladimir; Jiang, Xiaoxu et al. (2018) Oversized galactosides as a probe for conformational dynamics in LacY. Proc Natl Acad Sci U S A 115:4146-4151
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
Jiang, Xin; Smirnova, Irina; Kasho, Vladimir et al. (2016) Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation. Proc Natl Acad Sci U S A 113:12420-12425