NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F(1)F(0)-ATPase.
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.
Stelzer
Andrew C
AC
Department of Biophysics, University of Michigan, Ann Arbor, MI 48109, USA.
Frazee
Richard W
RW
Van Huis
Chad
C
Cleary
Joanne
J
Opipari
Anthony W
AW
Glick
Gary D
GD
Al-Hashimi
Hashim M
HM
D001569:Benzodiazepines / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D016503:Drug Delivery Systems; D007155:Immunologic Factors / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure
D007155:Immunologic Factors; D001569:Benzodiazepines; D025261:Mitochondrial Proton-Translocating ATPases; C468549:Bz-423