Nmr Studies Of An Immunomodulatory Benzodiazepine Binding To Its Molecular Target On The Mitochondrial F1f0-Atpase

Bz-423 is an inhibitor of the mitochondrial F1F0-ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water-soluble Bz-423 analog (5-(3-(amiriometliyl)phenyl)-7-chloro-1-mtethyl-3-(naphtltalen-2-ylttiethyl)-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 rising chemical shift perturbation and cross-relaxation experiments. Titration experiments with constructs representing residues 1-120 or 1-145 of the OSCP reveals that (a) I 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 I 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. (C) 2009 Wiley Periodicals, Inc. Biopolymers 93: 85-92, 2010.