The 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.

双功能蛋白 ACAD9 在脂肪酸 β-氧化过程中催化脂肪酰基辅酶 A 硫酯的 α,β-脱氢，是线粒体呼吸复合物 I (CI) 组装的重要伴侣。 ACAD9、ECSIT 和 NDUFAF1 相互作用形成核心线粒体 CI 组装复合体。目前的研究探讨了 ACAD9/ECSIT/NDUFAF1 相互作用的分子机制。 ACAD9 与 ECSIT 的羧基端一半结合，NDUFAF1 与 ECSIT 的氨基端一半结合。二元复合物不稳定且容易聚集，而ACAD9/ECSIT/NDUFAF1三元复合物可溶且高度稳定。分子建模和小角 X 射线散射研究确定了复合物内相互作用位点和其他组装因子的结合位点。 ECSIT 在 ACAD9 氨基末端结构域中 ETF 结合位点的结合与观察到的 FAD 和酶活性的损失一致，并证明 ACAD9 的两种功能是相互排斥的。将 42 个已知致病突变映射到同源模型 ACAD9 结构上，为 CI 缺陷的病理机制提供了结构见解。