Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b₅

Abstract

Cytochrome b5 (cyt b5) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt b5 modulation of CYP catalysis. Both cyt b5 and apo b5, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo b5 with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo b5 with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b5 and apo b5 were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt b5 binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b5 and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt b5 can dock with CYP3A4 in a manner analogous to holo cyt b5 so electron transfer from cyt b5 is not required for its effects.

Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5

“…The interaction sites of b 5 with CYP2B4 (33, 34), CYP2E1 (35), CYP3A4 (32), and CYP17A1 (this study and Refs. 6, 7) identified by cross-linking, NMR mapping, and site-directed mutagenesis, are summarized in Table 4.…”

“…The CYP17A1 mutation K88A shows a 30 -40% diminished affinity for b 5 (29,30), and CYP17A1 mutation K89N exhibits a selective 3-fold reduction in 17,20-lyase activity (31). In fact, Lys-88 of CYP17A1 corresponds to Lys-96 of CYP3A4, and the latter has been shown to interact with the same residue on helix 4 of b 5 (Glu-61, human b 5 numbering) (32). When Arg-347 of CYP17A1 located on helix JЈ was mutated to lysine, a cross-link was readily formed with Glu-42 of the b 5 molecule (Fig.…”

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

“…A biophysical and biochemical understanding of these critical proteinprotein interactions has been limited by the absence of crystallographic structures for human P450 proteins interacting with these protein partners, although mutagenesis, crosslinking, in silico docking, and other techniques have provided substantial information (Bridges et al, 1998;Naffin-Olivos and Auchus, 2006;Im and Waskell, 2011;Zhao et al, 2012). The generation of X-ray structures of such P450-protein complexes is likely hindered by the transient nature of these P450-protein interactions and the fact that they are largely thought to be mediated by electrostatic interactions; however, defining and characterizing such P450-protein interactions at high-resolution are possible using solution NMR, which generates information on protein dynamics and conformations but is amenable to the lower affinities of P450-protein interactions.…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…Indeed, similar intermolecular electrostatic interactions between P450s and their functionally relevant redox partners cytochrome P450 reductase (CPR) and cytochrome b 5 (b 5 ) have been established through site-directed mutagenesis and chemical cross-linking coupled LC-MS/MS analyses (57)(58)(59)(60). Thus, we posited that if such CYP3A4 negatively charged clusters were in fact to interact with positively charged residues of one or more proteins in each of these E2-E3 complexes, then such intermolecular electrostatic interactions would be disrupted in the presence of high salt concentrations in the incubation systems, thereby attenuating if not completely abrogating CYP3A4 ubiquitination.…”