• Institution: Max-Planck Gesellschaft MPDL
  • Research in the chemical sciences including biochemistry, inorganic chemistry, organic chemistry, and physical chemistry.
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Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

  1. Dimitrios Fotiadisa,b,2
  1. aInstitute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland;
  2. bSwiss National Centre of Competence in Research TransCure, University of Bern, CH-3012 Bern, Switzerland;
  3. cComputational Biomolecular Dynamics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany
  1. Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved July 26, 2016 (received for review April 4, 2016)

Significance

Disease-causing bacteria are able to survive the strongly acidic environment of the stomach by activating extreme acid-resistance responses. One of these responses in gut bacteria consists of converting l-arginine into agmatine, which results in removal of one proton from the cytoplasm. In Escherichia coli, the transport protein AdiC assures the efflux of agmatine in exchange with l-arginine. We have solved the structures of wild-type AdiC in the presence and absence of the substrate agmatine at high resolution, allowing for the identification of crucial water molecules and of their functional roles in the substrate-binding pocket. Furthermore, structure-based site-directed mutagenesis combined with a radioligand binding assay improved our understanding of substrate binding and specificity of the l-arginine/agmatine antiporter AdiC.

Abstract

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Footnotes

  • 1H.I. and J.-M.J. contributed equally to this work.

  • 2To whom correspondence should be addressed. Email: dimitrios.fotiadis{at}ibmm.unibe.ch.
  • Author contributions: B.L.d.G. and D.F. designed research; H.I., J.-M.J., V.G., and Z.U. performed research; H.I., J.-M.J., V.G., Z.U., B.L.d.G., and D.F. analyzed data; and H.I., J.-M.J., and D.F. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.wwpdb.org. (PDB ID codes 5J4N and 5J4I).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1605442113/-/DCSupplemental.

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