Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states.
To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.
Kihira
Yoshitaka
Y
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
Iwahashi
Akihiro
A
Majima
Eiji
E
Terada
Hiroshi
H
Shinohara
Yasuo
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003545:Cysteine / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology* / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D001278:Atractyloside; C033531:eosin maleimide; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine; C003853:carboxyatractyloside; D004801:Eosine Yellowish-(YS)