title: New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.
abstract: The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy.
authors.forename: Rosita
authors.initials: R
authors.lastname: Curcio
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy.
authors.forename: Luigina
authors.initials: L
authors.lastname: Muto
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: Ciro Leo
authors.initials: CL
authors.lastname: Pierri
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy; Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Montalto
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy.
authors.forename: Graziantonio
authors.initials: G
authors.lastname: Lauria
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Onofrio
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fiorillo
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Fiermonte
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Lunetti
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Vozza
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy. Electronic address: loredana.capobianco@unisalento.it.
authors.forename: Loredana
authors.initials: L
authors.lastname: Capobianco
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy. Electronic address: annarita.cappello@unical.it.
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Cappello
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy; Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy. Electronic address: vincenza.dolce@unical.it.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Dolce
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D015870:Gene Expression; D007656:Ketoglutaric Acids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; C093171:methanethiosulfonate ethylammonium; C021723:oxoglutarate translocator; D000226:Mitochondrial ADP, ATP Translocases; D001120:Arginine; D005020:Ethyl Methanesulfonate; D008239:Lysine; D003545:Cysteine
keywords: Comparative modeling; Oxoglutarate carrier; Site-directed mutagenesis; Structural rearrangement; Substrate translocation