Integrated approaches to symport mechanisms of membrane transporters PROJECT SUMMARY/ABSTRACT Our long-term objective is to understand the molecular mechanisms of cation/solute symport catalyzed by membrane carriers. These transporters play critical roles in maintaining normal cellular activities, are important in human health and disease, and can serve as drug targets and therapeutic delivery pathways. In this proposal, we plan to study the bacterial Na+-coupled melibiose permease (MelB), which utilizes energy stored in the electrochemical gradient of Na+, Li+, or H+ to drive the translocation of galactoside against its concentration gradient, and is a prototype for exploring molecular mechanisms of symporters in the MFS family that can use more than one cationic species for coupling. The MelB homologue expressed in blood-brain and blood-retina barriers catalyzes Na+-coupled uptake of docosahexaenoic acids (DHA)-carrying lysophosphatidylcholine (LPC), thus supplying essential DHA to brain and eyes for neural development and prevents neurodegeneration. For secondary-active transport in general, the coupling between the driving cation and cargo solute is obligatory, but the mechanisms underlying the energetic coupling remain largely unknown. We will elucidate the Na+-coupled symport mechanisms by a combined approach, including genetics, biochemistry, calorimetry, site-directed spin labeling (SDSL) with continuous-wave electron paramagnetic resonance spectroscopy (CW-EPRs), and 3-D X-ray crystallography. We have created high- affinity MelB-camelid single-domain nanobodies (Nbs) for crystallization of MelB, and have implemented isothermal titration calorimetry (ITC) measurements to determine the free-energy changes and heat capacity changes for the binding of MelB?s ligands (melibiose, Na+, Li+), alone or together, as well as the CW-EPRs to measure ligand-induced solvent accessibility changes and proximity changes. Based on our strong preliminary results, three independent but complementary aims are proposed to test our central hypothesis: the core of the symport mechanism is cooperative binding of co-substrates that induces the formation of an occluded intermediate state. Our integrated multi-disciplinary approach will provide important missing information into the cation/solute symport mechanisms and improve our fundamental knowledge of the ligand binding energetics and protein conformational changes in general, as well as directly impact on other studies of Na+- coupled transporters including the LPC transporter in brain and retina.

Public Health Relevance

Thisapplicationaimstoelucidatethemechanismsof cotransportbyanintegratedmulti-disciplinaryapproach. Success of the proposal will provide novel and essential structural and functional information for our mechanistic understanding of cation-coupled transporters, which will directly facilitate studies of transporters relatedtohumanhealthanddiseaseand mayhelpdevelopnoveldrugsforthepreventionandtreatmentofa broadrangeofneurodegenerativediseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Preusch, Peter
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Texas Tech University
Schools of Medicine
United States
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Hussain, Hazrat; Helton, Tyler; Du, Yang et al. (2018) A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability. Analyst 143:5702-5710
Hariharan, Parameswaran; Tikhonova, Elena; Medeiros-Silva, João et al. (2018) Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB. BMC Biol 16:85