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Experimental conditions can obscure the second high-affinity site in LeuT

Abstract

Neurotransmitter:Na+ symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na+-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na+ ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na+-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na+-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

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Figure 1: LeuT has two high-affinity leucine binding sites.
Figure 2: Impairment of leucine binding to the S2 site in LeuT.
Figure 3: Effect of detergent on LeuT binding activity.
Figure 4: Intact S1 and S2 sites are required for Na+-coupled transport by LeuT.

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Acknowledgements

We thank M. Miller-Cruz for the preparation of membranes, S. Stolzenberg for help with the FEP-MD simulations, and the Keck Biotechnology Resource Laboratory at Yale University for the quantitative amino acid analysis. This work was supported in part by US National Institutes of Health grants DA17293 and DA022413 (J.A.J.), U54GM087519 (H.W. and J.A.J.), DA12408 (H.W.) and DA023694 (L.S.).

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M.Q. designed, carried out and analyzed the functional characterization of LeuT. B.Z. expressed, purified and helped with the preparation of LeuT. J.A.J. helped design the functional characterization and, with L.S. and H.W., helped to interpret the data. All the authors participated in writing and editing the manuscript.

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Correspondence to Jonathan A Javitch.

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The authors declare no competing financial interests.

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Quick, M., Shi, L., Zehnpfennig, B. et al. Experimental conditions can obscure the second high-affinity site in LeuT. Nat Struct Mol Biol 19, 207–211 (2012). https://doi.org/10.1038/nsmb.2197

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