A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Abstract

Background: cytb 5 modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb 5 was solved by NMR, and the cytP450-binding site on cytb 5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb 5 -cytP450 complex is presented. Addition of a substrate strengthens the cytb 5 -cytP450 interaction. Significance: The cytb 5 -cytP450 complex structure will help unravel the mechanism by which cytb 5 regulates catalysis by cytP450.

Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis , in lipid bilayers

“…Close van der Waals interactions extend over the entire length of the helix pair, involving 11 residues from TM1 (at positions 31,32,35,36,38,39,42,43,45,46, and 49) and 9 residues from TM2 (at positions 74, 77, 78, 80, 81, 84, 87, 88, and 91), and contribute significantly to the tertiary structural stability (Fig. 4A).…”

“…The use of such bilayer preparations for supporting the native-like conformation of the M2 protein from Influenza A has been validated with the comparison of spectra from synthetic bilayers and from cellular membranes where the protein has been inserted by the cellular machinery and never removed from this environment or exposed to a detergent environment (28). Multiple recent membrane protein structures have now been determined by OS ssNMR (29)(30)(31)(32)(33)(34), and the first membrane protein structure has been obtained from MAS ssNMR (35). OS ssNMR generates information on the orientations of peptide planes with respect to the bilayer normal, and for a TM helix it yields the tilt angle of the helix relative to the lipid bilayer normal and rotational orientation about the helical axis along the entire length of helix.…”

Human Cytochrome P450 17A1 Conformational Selection

“…Other biophysical approaches (36 -38) support conformational heterogeneity but must be interpreted within the context of current static structures or that of molecular dynamics simulations whose predictions about molecular motions are not easily verifiable. Although NMR studies for soluble bacterial systems (20,39) and the smaller and more NMR-amenable b 5 protein (32,40) have begun to demonstrate the potential for NMR to provide a high resolution perspective on P450 conformation(s) free of crystallization constraints, protein NMR is a new approach to investigating membrane P450 conformational heterogeneity.…”

Differences in Methadone Metabolism by CYP2B6 Variants

“…Recently this variability was shown for the first time to extend to P450 variants, with some CYP1A2 mutants quite affected by b 5 (Palma et al, 2013). On the basis of several kinetic, mutagenesis, and NMR binding studies of wild-type rabbit CYP2B4, Waskell et al proposed a model for an electron transfer complex between the acidic convex surface of b 5 and the concave basic proximal surface of CYP2B4 (Im and Waskell, 2011;Ahuja et al, 2013). More specifically, the b 5 binding site is on the CYP2B4 C-helix and b-bulge, with Asp65 and Val66 of b 5 in contact with Arg122, Arg126, and Lys433 of CYP2B4, with Arg133 also critical for interaction of the two proteins.…”

“…More specifically, the b 5 binding site is on the CYP2B4 C-helix and b-bulge, with Asp65 and Val66 of b 5 in contact with Arg122, Arg126, and Lys433 of CYP2B4, with Arg133 also critical for interaction of the two proteins. Other sites, specifically Met137 and Lys139, which were important for CYP2B4 binding to b 5 , are thought to perturb the C-helix, and R422 also affected CYP2B4 binding (Ahuja et al, 2013). On the basis of sequence alignments, the CYP2B4 residues Arg122, Arg126, Met137, Lys139, and Arg422, which influence binding to b 5 , correspond to Lys122, Arg126, Met137, Lys139, and Lys422 of CYP2B6.…”