title: The mitochondrial citrate transport protein: evidence for a steric interaction between glutamine 182 and leucine 120 and its relationship to the substrate translocation pathway and identification of other mechanistically essential residues.
abstract: Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in > or =98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA.
authors.forename: Chunlong
authors.initials: C
authors.lastname: Ma
authors.forename: Sreevidya
authors.initials: S
authors.lastname: Remani
authors.forename: Rusudan
authors.initials: R
authors.lastname: Kotaria
authors.forename: June A
authors.initials: JA
authors.lastname: Mayor
authors.forename: D Eric
authors.initials: DE
authors.lastname: Walters
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Kaplan
mesh_terms: D019943:Amino Acid Substitution; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D008698:Mesylates; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; C020723:citrate-binding transport protein; D005973:Glutamine; C074502:methanethiosulfonate; D007930:Leucine