Coupled flexibility change in cytochrome P450cam substrate binding determined by neutron scattering, NMR, and molecular dynamics simulation

Abstract

Neutron scattering and nuclear magnetic resonance relaxation experiments are combined with molecular dynamics (MD) simulations in a novel, to our knowledge, approach to investigate the change in internal dynamics on substrate (camphor) binding to a protein (cytochrome P450cam). The MD simulations agree well with both the neutron scattering, which furnishes information on global flexibility, and the nuclear magnetic resonance data, which provides residue-specific order parameters. Decreased fluctuations are seen in the camphor-bound form using all three techniques, dominated by changes in specific regions of the protein. The combined experimental and simulation results permit a detailed description of the dynamical change, which involves modifications in the coupling between the dominant regions and concomitant substrate access channel closing, via specific salt-bridge, hydrogen-bonding, and hydrophobic interactions. The work demonstrates how the combination of complementary experimental spectroscopies with MD simulation can provide an in-depth description of functional dynamical protein changes.

Figure 1

(a) Superposed x-ray crystal structures of substrate-free (dark-gray/blue) and camphor-bound (light-gray/red) CYP101, depicting the five regions listed in Table 1 for which significant dynamic changes are observed between the two protein forms. The five regions are labeled individually in the camphor-bound form. (b) Comparison of neutron dynamic susceptibility measured over a broad energy range for the two protein forms. The wings of the susceptibility peak observed in the BASIS instrumental energy window are supplemented by the data taken from HFBS in the low-energy range and CNCS in the high-energy range. (c) Comparison of the dynamic susceptibility for the two protein forms calculated from MD simulations over the same energy range.

Figure 2

Comparison of backbone order parameters between substrate-free (dark-gray/blue) and camphor-bound CYP101 (light-gray/red) on a residue-by-residue basis. Order parameters were derived for the two protein forms using (a) experimental NMR data and (b) MD simulations.

Figure 3

Decomposition of total dynamic susceptibility of substrate-free (dark-gray/blue) and camphor-bound (light-gray/red) CYP101 into contributions from: (a) protein without the five regions listed in Table 1, (b) the five regions only, (c) methyl rotations in the five regions, and (d) the five regions with methyl rotations removed.

Figure 4

Average structures of (a) camphor-bound and (b) substrate-free CYP101 obtained from MD simulations colored by atomic root-mean-square fluctuations in a grayscale (color-scale online: 0 Å, blue; midpoint, white; and 1.2 Å, red). The structures are rotated from Fig. 1a so as to look down the I-helix for better viewing of the five regions. Time series of backbone traces of the five protein regions during 100-ns MD trajectories of (c) camphor-bound and (d) substrate-free CYP101 in a grayscale (color-scale online: 0 ns, blue; midpoint, white; and 100 ns, red). The closed 3L63 crystal structure is shown as reference. (e) Key salt-bridge, hydrogen-bonding, and hydrophobic interactions in CYP101, in which the five regions are shown similar to Fig. 1a.