The ability of the cell to tightly regulate the temporal and spatial movement of molecules across membranes is central to its survival. This movement has to be done in a selective manner to ensure that the chemistry of the cytoplasm and other internal compartments is not disturbed. To carry out these tasks, membranes are studded with transporters and channels that are often specific to particular cell types or organelles. The primary objective of the current proposal is to use computational methods to examine the conformational changes and functional operation of the sugar transporter vSGLT. vSGLT is the bacterial member of the solute sodium symporter family of transporters responsible for adsorption of simple sugars in the small intestine and kidneys of humans. vSGLT is related to a very large superfamily of transporters called the five helix inverted repeat (5HIR) superfamily. An increased understanding of their molecular workings has the potential to help in treating disease states related to type 2 diabetes mellitus (T2DM) and the treatment of severe dehydration.
In Aim 1, we will study the coupling of Na+ and sugar release into the cell. We hypothesize that Na+ exit allows blocking residues to move out of the way and allow sugar to escape, much like opening a gate with a key. All structures of 5HIR superfamily members exhibit these gates, so elucidating this step could be widely informative to other cotransporters. Computations in the Grabe lab will be aided by transport assays on mutant vSGLTs in the Abramson and Wright labs. Our goal in Aim 2 is to use computational drug discovery to design potent inhibitors to vSGLT and hSGLT2. hSGLT2 is a drug target for treating T2DM, so our efforts, coupled with screening in the Wright lab, could lead to new therapies. High-affinity inhibitors to vSGLT would provide a new tool for stabilizing and crystallizing the unknown, outward-facing structure of vSGLT.
In Aim 3, we will use transition path sampling coupled with GPU-accelerated dynamics to generate the ensemble of paths between the outward-facing and inward-facing conformations. These simulations will reveal, in molecular detail, the mechanical escapement that allows the 5HIR superfamily to move substrates in the presence of a Na+ gradient. These studies will be guided by experimental SAXS/WAXS and DEER measurements in the Abramson lab. Finally, hSGLT1 plays a central role in the treatment of severe dehydration through the use of Oral Rehydration Therapy, which is estimated to save 1-3 millions lives per year since its inception. Treatment + consists of ingestion of a glucose/NaCl solution. The glucose and Na are absorbed across the brush border membrane by hSGLT1 in the intestine and subsequently deposited in the blood. Each transported mole of glucose is accompanied by 4-6 L of water. We will determine how and in which state(s) vSGLT allows water to permeate, and we will explore the effect of different sugars on water permeation. This final set of computations may suggest improved solutions to aid in rehydration of severally dehydrated patients. 1

Public Health Relevance

This study is to determine how vSGLT transports galactose across the membrane by harnessing the energy stored in sodium gradients. The results of our studies on vSGLT will impact our understanding of closely related human transporters including those involved in energy metabolism, thyroid metabolism, and depression. Our docking and ligand free energy simulations have the potential to aid in the design of human hSGLT2 inhibitors to treat type 2 diabetes mellitus. 1

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM089740-02
Application #
8331575
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2011-09-30
Project End
2016-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$282,027
Indirect Cost
$85,770
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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