MIA40 and ALR of the MIA pathway mediate the import of protein precursors that form disulfides into the mitochondrial intermembrane space. This import pathway is suggested to be a linear pathway in which MIA40 first binds to the precursor via a disulfide linkage and oxidizes it. Subsequently, ALR re-oxidizes MIA40 and then ALR transfers electrons to terminal electron acceptors. However, the precise mechanism by which ALR and MIA40 coordinate translocation is unknown. With a collection of small molecule modulators (MB-5 to MB-9 and MB-13) that inhibit ALR activity, we characterized the import mechanism in mitochondria. NMR studies show that most of the compounds bind to a similar region in ALR. Mechanistic studies with small molecules demonstrate that treatment with compound MB-6 locks the precursor in a state bound to MIA40, blocking re-oxidation of MIA40 by ALR. Thus, small molecules that target a similar region in ALR alter the dynamics of the MIA import pathway differently, resulting in a set of probes that are useful for studying the catalysis of the redox-regulated import pathway in model systems.
Keywords: NMR spectroscopy; drug screening; mitochondrial import pathway.