Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

13-POS Board 4 PROTON-DRIVEN ALTERNATING ACCESS IN A SPINSTER TRANSPORTER, AN EMERGING FAMILY OF BROAD-SPECIFICITY EFFLUX PUMPS Reza Dastvan 1 ; Ali Rasouli 2,3 ; Sepehr Dehghani-Ghahnaviyeh 2,3 ; Samantha Gies 1 ; Emad Tajkhorshid 2,3 ; 1 Saint Louis University School of Medicine, Biochemistry and Molecular Biology, Saint Louis, MO, USA 2 University of Illinois at Urbana-Champaign, NIH Center for Macromolecular Modeling and Bioinformatics, Urbana, IL, USA 3 University of Illinois at Urbana-Champaign, Biochemistry, and Center for Biophysics and Quantitative Biology, Urbana, IL, USA Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. S1P regulates growth, survival and migration of cells with profound proangiogenic effects. In humans, Spns2 acts as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P export and signaling. Using an integrated approach in lipid membranes, we combined double electron-electron resonance spectroscopy with molecular dynamics simulations to characterize major conformational states of a close bacterial homolog of the Spns proteins from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for the protonation step of the cycle and its regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical, ligand-dependent alternating access, in the absence of an obvious outward- facing conformational state. While our inward-facing model is similar to the X-ray structure, we have detected and generated a novel occluded state of HnSpns in the membrane. A conserved periplasmic salt bridge (Asp60 TM2 -Arg289 TM7 ) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Decoupled transmembrane proton-dependent conformational changes highlight a distinctive feature of HnSpns, and potentially other Spns proteins, that may be facilitated by more flexible transmembrane region particularly the gating helices. Accordingly, a considerably lower periplasmic pK of conformational changes compared with the intracellular side implies the functional necessity of an inwardly directed proton gradient across the membrane. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity, underscoring a previously unknown role for Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling.

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