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bioRxiv · 10.1101/307710

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

Leone, V.·Waclawska, I.·Koshy, C.·Kossman, K.·Sharma, M.·Prisner, T. F.·Ziegler, C.·Endeward, B.·Forrest, L. R.
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Abstract

The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD (1), whereby the dynamics of the protein are minimally biased so as to reproduce the experimental data. Comparison of the magnitude of the biasing work required for different input structures permitted us to assign them to specific states of the transport cycle under each of the experimental conditions. In particular, this analysis showed that BetP adopts inward-facing conformations in the presence of excess sodium, and a mixture of states when betaine is added. These studies better delineate the major conformations adopted by BetP in its transport cycle, and therefore provide important insights into its mechanism. More broadly, we illustrate how integrative simulations can aid interpretation of ambiguous structural and spectroscopic data on membrane proteins.\n\nAbbreviations

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Leone, V., Waclawska, I., Koshy, C., Kossman, K., Sharma, M., Prisner, T. F., Ziegler, C., Endeward, B., Forrest, L. R.. 2018-04-27. Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations. https://doi.org/10.1101/307710

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