Solution Structural Ensembles of Substrate-Free Cytochrome P450_cam

Abstract

Removal of substrate (+)-camphor from the active site of cytochrome P450cam (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The 1H,15N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for 15N-1H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. NMR-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information regarding relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

“…2A). The broadening of some NMR peaks in both the Fe(III)-H 2 O and Fe(II)-CO spectra ruled out a paramagnetic effect as the underlying cause for broadening, suggesting that multiple conformations undergo slow exchange in the NMR time scale, as suggested previously for CYP101 (55). Apart from appearance of the N and O signals, the ligand-free CYP119 and Fe(II)-CO-CYP119 had very similar spectral properties, in agreement with their very similar crystal structures (47).…”

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

“…The different structures obtained for the substrate-free enzyme depending on whether the substrate is soaked out of crystals (23) or is absent during crystallization (26) suggest that crystal contacts might influence the conformational equilibrium and warn that the states seen in the crystal do not necessarily reflect those populated in solution. In addition, while a recently reported NMR study (28) has shown that camphor binding results in alteration of the conformational ensemble in solution, large-scale movements of the F and G helices were not indicated. It was suggested that crystal contacts may be responsible for this difference, but it is possible that the presence of CO in the NMR studies may also contribute to changing the conformational equilibrium.…”

“…However, these conformations were observed for the protein in the crystalline state, and information is currently lacking about the conformations that are actually populated in the physiologically relevant solution state. This issue is particularly important given the recent report of NMR studies (28), which demonstrate significant changes in the conformational ensemble upon camphor binding, but did not reveal large movements in the F and G helices. Therefore, it has become critically important to establish whether the previously reported open conformations are seen in the solution state.…”

Human Cytochrome P450 17A1 Conformational Selection

“…Like most P450 enzymes, the long protein-spanning I helix of CYP17A1 has a noncanonical hydrogen bonding arrangement just as it passes over the heme. In existing structures of some P450 enzymes (14,23,39), this results in an I helix that has been observed in either a straight conformation or with a kink arising at the midpoint, allowing movement of the N-terminal half of the I helix. In CYP17A1, the carbonyl of Ala-302 clearly hydrogen-bonds to the side chain hydroxyl of Thr-306 rather than the Thr-306 backbone.…”