RESUMO
Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.
Assuntos
Fator de Indução de Apoptose , Domínio Catalítico , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais , Modelos Moleculares , Ligação Proteica , Fator de Indução de Apoptose/metabolismo , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Regulação Alostérica , Cristalografia por Raios X , NAD/metabolismo , NAD/química , Sítios de Ligação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
To perform their action, flavoproteins usually interact with a variety of low molecular weight partners, including electron transporters, yielding transient complexes whose tightness is often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe the quantitative analysis of the redox-dependent interaction of the mammalian apoptosis inducing factor (AIF) with its NAD+ ligand. In particular, we report a protocol for the spectrophotometric titration of AIF in its reduced state under anaerobic conditions with NAD+, in order to determine the dissociation constant of the resulting complex.
Assuntos
Fator de Indução de Apoptose/metabolismo , Escherichia coli/crescimento & desenvolvimento , NAD/metabolismo , Regulação Alostérica , Anaerobiose , Animais , Fator de Indução de Apoptose/genética , Escherichia coli/genética , Camundongos , Proteínas Recombinantes/metabolismo , EspectrofotometriaRESUMO
To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.
