title: Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.
abstract: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.
authors.affiliation: Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Sorrentino
authors.affiliation: Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
authors.forename: Alessandra Maria
authors.initials: AM
authors.lastname: Calogero
authors.affiliation: Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Pandini
authors.affiliation: Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Vanoni
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California , Irvine, California 92697-3900, United States.
authors.forename: Irina F
authors.initials: IF
authors.lastname: Sevrioukova
authors.affiliation: Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
mesh_terms: D000818:Animals; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D051379:Mice; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D013696:Temperature
chemical_list: D051033:Apoptosis Inducing Factor; D024101:Mitochondrial Proteins; C494830:AIFM1 protein, mouse; D009243:NAD