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.

Dimers of mitochondrial ATP synthase form the permeability transition pore

“…S1C. This region overlaps with helices 3 and 4, the binding site of benzodiazepine 423 (Bz-423), a wellcharacterized inhibitor of the F O F 1 ATP synthase that readily permeates mitochondria (27,28). We, therefore, tested the effect of Bz-423 on the association of CyPD to mouse and bovine complex V at 10 mM Pi, and we found a concentrationdependent displacement that is consistent with competition for a common binding site (Fig.…”

“…Bz-423 was discovered and characterized as an apoptosis-inducing agent acting through mitochondria (39); identification of OSCP as its target was achieved through the screening of a human cDNA T7 phage display library (27) and the interaction with ATP synthase resulting in inhibition of enzyme activity confirmed by NMR (28). The striking selectivity of action of Bz-423 on OSCP and its ability to trigger channel activity of ATP synthase dimers, together with the lack of activity of monomer preparations, argues against the possibility that the currents that we observe are caused by unidentified contaminating proteins.…”

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

“…For example, these studies have identified the site of Bz-423 binding on the OSCP subunit of this complex. Bz-423 both promotes PTP opening (37) and inhibits the F 0 F 1 -ATP synthase (66,72). Moreover, many "TSPO ligands" also bind to and inhibit the mitochondrial F O F 1 -ATP synthase (66, 70 -72), and this interaction probably best explains their effects in cell death and autophagy (73) through an inducing effect on PTP formation by ATP synthase dimers (37).…”

From ATP to PTP and Back

“…127 As already mentioned, this subcomplex is also the target of CyPD, which binds to the OSCP subunit (Figure 2), possibly sharing a common binding site with the F-ATP synthase inhibitor Bz-423. 128,129 CyPD binding to OSCP is favored by Pi and counteracted by CsA; 77 CyPD sensitizes the PTP to Ca 2+ , promoting the transition of the F-ATP synthase to a channel that may form at the interface between monomers 51–53,61 (Figure 2). Interestingly, OSCP has also been recently recognized as the binding site of 17β-estradiol, whose binding promotes an intrinsically uncoupled state of ATP synthase, 130 as well as of regulatory proteins, such as Sirtuin3, 131 highlighting its ability to sense metabolic signals.…”