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

Abstract

ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate 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 modelling, to arrive at a topological model for ArgO. Our studies indicate that ArgO assumes an N_in-C_out configuration potentially forming a five transmembrane helix bundle flanked by an N-terminal cytoplasmic domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short C-terminal periplasmic 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, implicate a role for intramolecular interactions in the Arg export mechanism and some interactions are corroborated by normal modes analyses. Lastly our studies suggest that in vivo ArgO may exist as a monomer, highlighting thus 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, L-lysine and L-arginine respectively, and LysE can functionally substitute ArgO when expressed in E. coli. Notwithstanding this functional equivalence, studies herein 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.