Structural basis for substrate binding and specificity of a sodium–alanine symporter AgcS

Jinming Ma, Hsiang-Ting Lei, Francis E. Reyes, Silvia Sanchez-Martinez, Maen F. Sarhan, Johan Hattne, and Tamir Gonen
PNAS February 5, 2019 116 (6) 2086-2090; published ahead of print February 5, 2019 https://doi.org/10.1073/pnas.1806206116

  1. Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved December 12, 2018 (received for review April 11, 2018)

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Significance

The substrate binding and specificity are essential features for the amino acid–polyamine–organocation superfamily members. Here, we report two crystal structures of an alanine transporter, alanine or glycine:cation symporter, capable of transporting both L- and D-alanine and discovered that both the size and polarity of the substrate binding pocket are important structural determinants for specificity and stereo-selectivity. The size of the binding pocket determines what amino acids could fit in the transporter, while electrostatics control whether L-type or D-type amino acids could bind. Mutations in the binding site can broaden the specificity of this transporter to allow uptake of larger amino acids, such as valine and leucine.

Abstract

The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: tgonen{at}ucla.edu.

  • Author contributions: T.G. designed research; J.M., H.-T.L., F.E.R., S.S.-M., and M.F.S. performed research; J.M., H.-T.L., F.E.R., S.S.-M., M.F.S., J.H., and T.G. analyzed data; and J.M., H.-T.L., M.F.S., J.H., and T.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (Protein Data Bank ID codes 6cse and 6csf).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1806206116/-/DCSupplemental.

Published under the PNAS license.

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Structural basis for substrate binding and specificity of a sodium–alanine symporter AgcS
Jinming Ma, Hsiang-Ting Lei, Francis E. Reyes, Silvia Sanchez-Martinez, Maen F. Sarhan, Johan Hattne, Tamir Gonen
Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2086-2090; DOI: 10.1073/pnas.1806206116
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