title: Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups.
abstract: The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [(3)H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.
authors.affiliation: Department of Biosciences, Biotechnology and Pharmacological Sciences, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Tonazzi
authors.forename: Lara
authors.initials: L
authors.lastname: Console
authors.forename: Nicola
authors.initials: N
authors.lastname: Giangregorio
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
mesh_terms: D000215:Acylation / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D002331:Carnitine / Q000031:analogs & derivatives / Q000494:pharmacology; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects*; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010172:Palmitoylcarnitine / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D050505:Mutant Proteins; C116917:acylcarnitine; D010172:Palmitoylcarnitine; D002333:Carnitine Acyltransferases; D002331:Carnitine