Experimentally Restrained Molecular Dynamics Simulations for Characterizing the Open States of Cytochrome P450_cam

Abstract

Residual dipolar couplings were used as restraints in fully solvated molecular dynamics simulations of reduced substrate-and carbonmonoxy-bound cytochrome P450 cam (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The 1 D NH residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site, and largely support previously reported differences between the open and closed states of CYP101A1.The sine qua non of enzyme function is that the three-dimensional structure of an enzyme stabilizes the transition state of the reaction being catalyzed (1). This implies that the enzyme structure cannot stabilize the reactant (or product) ground states, at least not at the point of catalysis. However, enzymes have another job to do as well: They must concentrate reactants in the active site, that is, the thermodynamics of substrate binding must be sufficiently favorable so that substrate is bound selectively and with reasonable affinity. How then to square this particular circle? One way is to assume an enzyme is able to access different conformations in order to accomplish both tasks (2). If this is true, one would expect to find accessible "open" enzyme conformers that allow substrate access and bind substrate with a favorable free energy, and a different set of "closed" conformers that stabilize the appropriate transition states for catalysis. Furthermore, the activation barriers separating the open and closed conformers should be sufficiently high that each a This work was supported in part by a grant from the USPHS (R01-GM44191, TCP). EKA and JDM acknowledge support from USPHS grant R01DA027806, a Department of Education grant P116Z090309 and NSF support through Teragrid (MCB060061N). b The REP structure discussed in this paper has been deposited as PDB entry 2l8m. NMR chemical shifts and RDC restraints are deposited as BMRB entry 17415. * Inquiries should be addressed to this author at Dept. of Chemistry, MS 015, Brandeis University, 415 South St., Waltham, MA 02454-9110. Tel.: 781-736-2559, fax: 781-736-2516, pochapsk@brandeis.edu, website: www.chem.brandeis.edu/pochapsky. Supplementary Material Available: PDB format structure file for REP structure, RDC restraints in AMBER format and two tables discussed in the text are available in online at http://pubs.acs.org. conformation is discretely accessible on the time scale of catalysis, but not so formidable as to preclude access to either. NIH Public AccessWe have used multidimensional nuclear magnetic resonance (NMR) sp...

Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins

“…The second Leu16 conformation is associated with a smaller rotation of the 7-propionate. Significantly, this conformer exhibits a significantly bent Fe-C-O unit with a bond angle of 158 AE 8°( Table 1) comparable to that found in a variety of haemoproteins at atomic resolution structures (7)(8)(9)(10)(11)(12).…”

Solution Conformations and Dynamics of Substrate-Bound Cytochrome P450 MycG

“…RDC restraints were then introduced gradually with protein atoms free to move, with penalties increased from 0.001 to 0.01 kcal mol −1 (δD) −2 over several nanoseconds of simulation at 300 K, where δD is the difference between the observed and calculated RDC exceeding the +/− 3 Hz uncertainty of measurement for a given restraint. The final penalty value used for production runs (0.01 kcal mol −1 (δD) −2 ) was found in our previous work to impose a reasonable restraint set without distortion of local geometry (14). Runs were halted every 0.25 nsec in order to detect and correct any significant violations of RDC restraints, either due to misassignment or incorrect measurement.…”

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A novel type of allosteric regulation: Functional cooperativity in monomeric proteins

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