Linkage between Proximal and Distal Movements of P450cam Induced by Putidaredoxin

Abstract

Putidaredoxin (Pdx) is the exclusive reductase and a structural effector for P450cam (CYP101A1). However, the mechanism of how Pdx modulates the conformational states of P450cam remains elusive. Here we report a putative communication pathway for the Pdxinduced conformational change in P450cam using results of double electron−electron resonance (DEER) spectroscopy and molecular dynamics simulations. Use of solution state DEER measurements allows us to observe subtle conformational changes in the internal helices in P450cam among closed, open, and P450cam−Pdx complex states. Molecular dynamics simulations and dynamic network analysis suggest that Pdx binding is coupled to small coordinated movements of several regions of P450cam, including helices C, B′, I, G, and F. These changes provide a linkage between the Pdx binding site on the proximal side of the enzyme and helices F/G on the distal side and the site of the largest movement resulting from the Pdx-induced closed-toopen transition. This study provides a detailed rationale for how Pdx exerts its long-recognized effector function at the active site from its binding site on the opposite face of the enzyme.

Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase

“…46 This was first explored with P450cam, whose association with the electron donor (Pdx) was found to cause a shift from a closed to an open conformation of the P450 enzyme via motions of the Fand G-helices. 8,9 A putative communication pathway has been proposed for the Pdx-induced conformational change via coupled motions of the B'-, C-, I-, D-and F-helices, 56 which is consistent with our results on the CYP3A4-CPR pair (Figures 1 and S8). Among all regions of CYP3A4 impacted by CPR binding, the structural rigidification was strongest in the B'-helix, which defines two major substrate access channels, and the active site K/1-loop, which participates in substrate binding.…”

“…CPR is believed to undergo a drastic conformational change upon reduction. 2–4,56 The reduced form of CPR ( red CPR) was also reported to have a higher affinity for CYPs than ox CPR. 43 Therefore, we hypothesized that binding of red CPR might affect the structural dynamics of CYP3A4 differently than ox CPR.…”

“…32 Conformational flexibility in the I-helix was also observed in P450cam and thought to be important for oxygen activation and binding of the redox partner Pdx. 8,[56][57][58] These data illustrate the potential for CPR binding to modulate the structural dynamics of CYP3A4 with a finely-tuned regioselectivity that depends on the redox state of the reductase.…”

“…During these electron transfer steps, CPR undergoes large conformational changes from an open to a closed form. [2][3][4]56 In addition, various studies have reported that CPR may induce conformational changes in CYPs that promote inter-protein electron transfer. 6 However, the current understanding of conformational changes induced by redox partners on CYPs is based mostly on the bacterial P450cam-putidaredoxin (Pdx) system.…”

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding

“…Additionally, basic residues on the proximal side of CYPs seem to steer the negatively charged convex binding-interface of the FD to CYP’s concave docking site near the heme moiety [ 9 ]. Interestingly, structural information on CYP substrate/ligand complexes and conformational dynamics for substrate access demonstrated that CYP’s active site adapts to substrates of diverse sizes and shapes, which in turn may have implications in the CPR:CYP interaction as the structural rearrangement associated with substrate binding was shown to extends its effects to the proximal side of CYPs [ 1 , 2 , 27 , 28 , 29 , 30 , 31 ].…”