摘要：SummaryThe dual function protein ACAD9 catalyzes α,β-dehydrogenation of fatty acyl-CoA thioesters in fatty acid β-oxidation and is an essential chaperone for mitochondrial respiratory complex I (CI) assembly. ACAD9, ECSIT, and NDUFAF1 interact to form the core mitochondrial CI assembly complex. Current studies examine the molecular mechanism of ACAD9/ECSIT/NDUFAF1interactions. ACAD9 binds to the carboxy-terminal half and NDUFAF1 to the amino-terminal half of ECSIT. Binary complexes are unstable and aggregate easily, while the ACAD9/ECSIT/NDUFAF1 ternary complex is soluble and highly stable. Molecular modeling and small-angle X-ray scattering studies identified intra-complex interaction sites and binding sites for other assembly factors. Binding of ECSIT at the ETF binding site in the amino-terminal domain of ACAD9 is consistent with observed loss of FAD and enzymatic activity and demonstrates that the two functions of ACAD9 are mutually exclusive. Mapping of 42 known pathogenic mutations onto the homology-modeled ACAD9 structure provides structural insights into pathomechanisms of CI deficiency.Graphical abstractDisplay OmittedHighlights•ECSIT bridges ACAD9 and NDUFAF1 in the ACAD9/ECSIT/NDUFAF1 heterohexamer of MCIA•The NH2-domain of ECSIT binds to NDUFAF1, whereas the COOH-domain binds to ACAD9•ECSIT binds to ACAD9 near the FAD binding site and deflavinates ACAD9•Dehydrogenase and complex 1 assembly functions of ACAD9 are mutually exclusiveBiological sciences; Molecular biology; Structural biology
