Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1)

Abstract

Cytochrome P450_cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide ¹H-¹⁵N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.

Keywords: anisotropic network model; cytochrome P450 stereospecificity; perturbation response scanning; residual dipolar couplings; solution conformational ensembles.

Figure 1

Camphor orientation in the active site of reduced and carbonmonoxy-bound CYP101A1 from crystal structure 3CPP [35]. Distance shown by dotted line (2.0 Å) is from the 5-exo-hydrogen of camphor to the carbon atom of bound carbon monoxide (approximately the same position as the active oxygen the Fe=O complex).

Figure 2

Solution structure 2L8M of CYP101A1 [5], with secondary structural features referred to in the text labeled according to the scheme of Poulos [35]. Heme is shown as red sticks, camphor as magenta. Residue numbers are as follows: A helix, V38-E47; β1, L53-A65; B helix, R67-D77; B′ helix, P89-E94; C helix, Q108-V119; D helix, D125-R143; E helix, F150-G168; F helix, P170-T185; G helix, T192-K214; H helix, A219-A224; β2, Q227-P232; I helix, T234-A265; J helix, S267-R277; K helix, R280-F292; β3, G298-L301 and Q317-L319; β4, Y305-L312; K′ helix P321-L327; β meander, D328-L358, L helix, Q360-I378; β5, S382-V405.

Figure 3

Improper angle Ψ defined between heme Fe, NB, camphor C7 and C9 for restraint of camphor orientation in the active site of CYP101A1.

Figure 4

Free energy profile extracted from umbrella sampling starting from 2L8M, with the reaction coordinate being the change in the improper dihedral angle Ψ as defined in Figure 3.

Figure 5

Effective force constant (K_ij) map for 2L8M in arbitrary units. Secondary structural features are labeled at the top of the map as in Figure 1.

Figure 6

Heat maps of Cα perturbation response scanning (PRS) of RDC-derived solution structures of CYP101A1 with (2L8M) and without (2LQD) d-camphor bound. The stronger the motional correlation, the “hotter” the cross-peak between positions. The most correlated regions in each map are labeled with reference to secondary structural features from Fig. 2.

Figure 7

Comparison of Cα PRS heat maps between unrestrained Ψ angle (left) and Ψ restrained to the starting angle from 2L8M after equilibration (88°). See Figure 2 for definition of Ψ. Axes correspond to residue number in both dimensions. Lettering corresponds to structural features shown in Figure 1. The intense feature labeled β5 in both maps corresponds to Gly 386 near the end of one strand of the β5 sheet.

Figure 8

Hydrogen bonding network involving Ser 346 (β-meander). Ser 346 is sensitive to motions in the B–B′ and B′–C loop in the presence of substrate (Fig. 7). The backbone amide and carbonyl of Ser 346 forms β-type hydrogen bonds with the side chain of Asn 332 (red dotted lines), which in turn is within hydrogen bonding distance of Ser 325 C=O in the K′ helix. The amide NH resonance of Ser 325 is strongly perturbed upon substrate binding [11], and Glu 331 was found by directed evolution to affect substrate selectivity in CYP101A1 (Ref. [16]).

Figure 9

PRS matrices for selected camphor orientations. Ψ= 26° (left), Ψ= −60° (center), and Ψ= −100° (right).

Figure 10

Left: Sensitivity profile in arbitrary units corresponding to the dihedral angle ψ= −140° (black line), compared with sensitivity profile with no restrictions applied to camphor (blue line). Right: position of Pro 215 at the end of the G helix, showing an increased sensitivity when ψ= −140°.

Figure 11

PRS heat map obtained for 2L8M with RDC restraints applied (right) compared with the map for unrestrained 2L8M (from Figs. 6 and 7).

Figure 12

Left, superposition of 2L8M on structures extracted from dynamics tracks with restraints on Ψ. Right, close-up of camphor orientations from the superposition: 2L8M (green), Ψ = 26° (cyan), Ψ −100° (salmon), Ψ = −60° (yellow).

Figure 13

First sphere contacts in CYP101A1 active site as a function of Ψ. Top left: 2L8M. Top right, R2: Ψ = 26 °. Bottom left, R3, Ψ = −60 °, bottom right Ψ = −100°. Methyl carbon C8 in substrate camphor is identified. In 2L8M, C8 contacts L244 and V247 on the I helix. In R2, C8 contacts V247, M184 and T185 (F). In R3, D297 is only major contact for C8, although F87 (B–B′) is within 5 Å. In R4, C8 contacts V295, D297 (β3) and I395 (β5).

Scheme 1