NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F1F0‐ATPase

Abstract

Bz-423 is an inhibitor of the mitochondrial F(1)F(0)-ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water-soluble Bz-423 analog (5-(3-(aminomethyl)phenyl)-7-chloro- 1-methyl-3-(naphthalen-2-ylmethyl)-1H-benzo][e][1,4]diazepin-2(3H)-one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross-relaxation experiments. Titration experiments with constructs representing residues 1-120 or 1-145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP-F(1) interface resulting in disrupted communication between the peripheral stalk and the F(1)-domain of the enzyme.