Conformational dynamics of substrate in the active site of cytochrome P450 BM-3/NPG complex: insights from NMR order parameters.

Cytochrome P450 enzymes catalyze selective oxidation of a wide variety of hydrophobic substrates.1 The low-temperature crystal structure P450 BM-32 bound to N-palmitoylglycine (NPG) shows the substrate bound distant to the iron in a position that is evidently unproductive for chemistry. UV-vis absorbance measurements indicating a spin-state transition and associated studies3 of cytochrome P450 BM-3 in complex with N-palmitoylglycine (NPG) suggest that a conformational change occurs in the active site of the complex where the terminal atoms (which undergo oxidation) of the ligand move from a site distant from the heme iron, as seen in the low-temperature crystal structure to a site proximal to the heme iron at biological temperatures. Prediction of this productive binding mode would be very useful for prediction and control of drug metabolism. We have carried out replica exchange molecular dynamics (REMD) simulations4 aimed at modeling the structural and dynamic properties of the productive, proximal complex.5 The population of the proximal state was found to increase with temperature in agreement with UV-vis absorbance and NMR measurements.3 In addition to conformations characterized by X-ray crystallography and computer modeling, this study5 showed that a new conformational state, which is stabilized by conformational entropy is significantly populated at room temperature. Information on fast internal motion of the protein contained in NMR relaxation experiments, can be specified by an effective correlation time and a generalized order parameter S.6-11 These quantities can be extracted from relaxation data using a fitting procedure. Order parameters8,12-14 can also be calculated from MD simulations,15 and here we use REMD trajectories of the P450 BM3/NPG simulations5 to calculate S2 for bond vectors of NPG and compare them to experimentally determined order parameters. This is the first time that NMR order parameters have been calculated from REMD simulations to study the conformational dynamics of a ligand in the active site of a protein. Free energy barriers between conformations prevent standard MD sampling often from achieving an equilibrium distribution of bond orientations required for order parameters, obtained from solidstate NMR line shape analysis.14 REMD4 is an advanced sampling algorithm that allows crossing of these barriers and facilitates the sampling of a more complete distribution of bond-vector orientations and corresponding order parameters. We have calculated the order parameters, shown in Figure 1, for the C-C bond vectors of N-palmitoylglycine at 294 K from an REMD simulation of P450 BM-3 with 24 replicas and an aggregate simulation time of 72 ns starting from the 1JPZ PDB crystal structure.5 The value of S2 for the terminal C-C bond (bond 17) at 294 K is 0.15. This is within the experimental range of 0.120.32 (the bar in Figure 1) reported by Jovanovic et al.14 based on solid-state line-shape analysis. The order parameters were dissected by averaging exclusively over the distal state (green line in Figure 1) and the proximal state (blue line in Figure 1). The value of the calculated order parameter S2 for bond 17 at 294 K corresponding to the proximal state is within the experimentally determined range, whereas the value of the calculated order parameter corresponding to the distal state is outside the experimentally determined range. The calculated order parameters for the proximal and distal states are consistent with the hypothesis that at room temperature the P450BM3/NPG complex is predominantly in the proximal state. The order parameters for all C-C bonds for the proximal state are smaller than those for the distal state (compare red and green lines in Figure 1). This implies that the distribution of orientations of bond vectors for the proximal state is wider than that for the distal state. Also the order parameters obtained by averaging exclusively over the proximal state (blue line in Figure 1) is approximately the same as that obtained by complete averaging over both proximal and distal states (red line in Figure 1). This implies that the distribution of orientations of bond vectors corresponding to the distal state overlaps significantly with the distribution of orientations corresponding to the proximal state. If the distribution of bond orientations of distal and proximal states had been very different and the region covered by them did not overlap, the order parameters obtained by averaging over both proximal and distal states, would have been much lower than that of the proximal and of the distal states separately. This is confirmed by the observed distribution of bond orientations for the representative bond 17 shown in Figure 2. The distribution corresponding to the proximal state (blue crosses) covers a significantly larger space as compared to the distal state (green crosses). The wider distribu† The State University of New Jersey. ‡ Columbia University. Figure 1. Order parameters at 294 K as a function of bond index of NPG by averaging over the whole trajectory (red), over exclusively the proximal state (blue), and exclusively the distal state (green). Lower indices correspond to bonds near the carboxylate end of NPG. Index 17 corresponds to the terminal C-C bond farthest from the carboxylate end of NPG and closest to the heme group of the protein. The range of order parameters extracted from experiments11 at room temperature for bond 17 is represented by the bar on the right (black). Published on Web 12/29/2006