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The Topology of the L-Arginine Exporter ArgO Conforms to an N-in-C-out Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Pathania, Amit and Gupta, Arvind Kumar and Dubey, Swati and Gopal, Balasubramanian and Sardesai, Abhijit A (2016) The Topology of the L-Arginine Exporter ArgO Conforms to an N-in-C-out Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function. In: JOURNAL OF BACTERIOLOGY, 198 (23). pp. 3186-3199.

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Official URL: http://dx.doi.org/10.1128/JB.00423-16

Abstract

ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of L-arginine (Arg) in Escherichia coli and L-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an N-in-C-out configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit. IMPORTANCE The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids L-arginine and L-lysine and the basic amino acid L-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic and in silico studies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

Item Type: Journal Article
Additional Information: Copy right for this article belongs to the AMER SOC MICROBIOLOGY, 1752 N ST NW, WASHINGTON, DC 20036-2904 USA
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Depositing User: Id for Latest eprints
Date Deposited: 30 Dec 2016 05:56
Last Modified: 30 Dec 2016 05:56
URI: http://eprints.iisc.ac.in/id/eprint/55595

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