Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism.

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.

Molecular modeling of O6-methylguanine-DNA methyltransferase mutant proteins encoded by single nucleotide polymorphisms.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) acts as a chemoprotectant and mediates resistance to alkylating anti-tumor agents. A number of MGMT single nucleotide polymorphisms (SNPs) have been described. We analyzed by molecular modeling the regions likely to be affected in the MGMT mutant proteins encoded by SNPs. Starting from the crystal structure of non-alkylated MGMT, molecular models of mutant proteins encoded by SNPs have been built. Most of the mutations were found to be located either within the DNA binding region (A121E, A121T, G132R, N123V) or in the vicinity of the active Cys145 (I143V, G160R). A further L84F mutant might affect Zn2+ binding. W65C was found to possibly be unstable.

Nonuniform charge scaling (NUCS): a practical approximation of solvent electrostatic screening in proteins.

In molecular mechanics calculations, electrostatic interactions between chemical groups are usually represented by a Coulomb potential between the partial atomic charges of the groups. In aqueous solution these interactions are modified by the polarizable solvent. Although the electrostatic effects of the polarized solvent on the protein are well described by the Poisson--Boltzmann equation, its numerical solution is computationally expensive for large molecules such as proteins. The procedure of nonuniform charge scaling (NUCS) is a pragmatic approach to implicit solvation that approximates the solvent screening effect by individually scaling the partial charges on the explicit atoms of the macromolecule so as to reproduce electrostatic interaction energies obtained from an initial Poisson--Boltzmann analysis. Once the screening factors have been determined for a protein the scaled charges can be easily used in any molecular mechanics program that implements a Coulomb term. The approach is particularly suitable for minimization-based simulations, such as normal mode analysis, certain conformational reaction path or ligand binding techniques for which bulk solvent cannot be included explicitly, and for combined quantum mechanical/molecular mechanical calculations when the interface to more elaborate continuum solvent models is lacking. The method is illustrated using reaction path calculations of the Tyr 35 ring flip in the bovine pancreatic trypsin inhibitor.

Protein/ligand binding free energies calculated with quantum mechanics/molecular mechanics.

The calculation of binding affinities for flexible ligands has hitherto required the availability of reliable molecular mechanics parameters for the ligands, a restriction that can in principle be lifted by using a mixed quantum mechanics/molecular mechanics (QM/MM) representation in which the ligand is treated quantum mechanically. The feasibility of this approach is evaluated here, combining QM/MM with the Poisson-Boltzmann/surface area model of continuum solvation and testing the method on a set of 47 benzamidine derivatives binding to trypsin. The experimental range of the absolute binding energy (DeltaG = -3.9 to -7.6 kcal/mol) is reproduced well, with a root-mean-square (RMS) error of 1.2 kcal/mol. When QM/MM is applied without reoptimization to the very different ligands of FK506 binding protein the RMS error is only 0.7 kcal/mol. The results show that QM/MM is a promising new avenue for automated docking and scoring of flexible ligands. Suggestions are made for further improvements in accuracy.

How well does charge reparametrisation account for solvent screening in molecular mechanics calculations? The example of myosin.

The rate constant of an enzyme-catalysed reaction is one of the major target properties to understand protein function. Atomic-detail computer simulations can in principle be used to estimate rate constants from the energy profile along the reaction coordinate. For such simulations, molecular mechanics is combined with a quantum description of the reaction process. In molecular mechanics calculations, the electrostatic field is represented by the Coulomb potential of partial atomic charges which have been parametrised for small building blocks in vacuum and transferred to the macromolecule. In aqueous solution, however, the electrostatic interactions are affected by the solvent polarization. While this can be described by numerically solving the Poisson-Boltzmann equation, it is computationally expensive. A simple approximation to this is to optimally reproduce the electrostatic potential in solution by reparametrising the partial atomic charges in such a way that a simple Coulomb potential can still be used. Such a procedure would allow to perform fast calculations of reaction processes in proteins while accounting for the solvent screening effect. Here, this method is tested on myosin, a motor protein that is both an enzyme and exists in very different conformations.

Time-resolved computational protein biochemistry: solvent effects on interactions, conformational transitions and equilibrium fluctuations.

Solvent plays an important role in modulating internal motions of proteins. Here we present a computational method for including solvent effects on charge-charge interactions and on pathways between functional protein conformations, and examine solvent effects on equilibrium internal fluctuations in proteins. A computationally efficient charge reparametrisation method is presented that satisfactorily reproduces the electrostatic interactions present in a full continuum Poisson-Boltzmann representation. The application of charge reparametrisation in the calculation of a large-scale conformational transition pathway in a protein, annexin V, is illustrated. We also examine solvent effects on fast (picosecond timescale) internal protein dynamics. Nosé-Hoover dual heatbath molecular dynamics simulations are performed. These simulations allow the solvent region to be fixed at one temperature and the protein at another. The results of the Nosé-Hoover simulations on hydrated myoglobin confirm that the solvent temperature strongly influences the protein fluctuations. We consider to what extent the solvent can be considered to determine the high temperature protein dynamics.

Computational tools for analysing structural changes in proteins in solution.

Many important structural changes in proteins involve long-time dynamics, which are outside the timescale presently accessible by a straightforward integration of Newton's equations of motion. This problem is addressed with minimisation-based algorithms, which are applied on possible reaction pathways using atomic-detail models. For reasons of efficiency, an implicit treatment of solvent is imperative. We present the charge reparameterisation protocol, which is a method that approximates the interaction energies obtained by a numerical solution of the Poisson-Boltzmann equation. Furthermore, we present a number of methods that can be used to compute possible reaction pathways associated with a particular conformational change. Two of them, the self-penalty walk and the nudged elastic band method, define an objective function, which is minimised to find optimal paths. A third method, conjugate peak refinement, is a heuristic method, which finds minimum energy paths without the use of an explicit objective function. Finally, we discuss problems and limitations with these methods and give a perspective on future research.

Can the calculation of ligand binding free energies be improved with continuum solvent electrostatics and an ideal-gas entropy correction?

The prediction of a ligand binding constant requires generating three-dimensional structures of the complex concerned and reliably scoring these structures. Here, the scoring problem is investigated by examining benzamidine-like inhibitors of trypsin, a system for which errors in the structures are small. Precise and consistent binding free energies for the inhibitors are determined experimentally for this test system. To examine possible improvement of scoring methods, we test the suitability of continuum electrostatics to account for solvation effects and use an ideal-gas entropy correction to account for the changes in the degrees of freedom of the ligand. The small observed root-mean-square deviation of 0.55 kcal/mol of the calculated relative to the experimental values indicates that the essentials of the binding process have been captured. Even though all six ligands make the same salt bridge and H-bonds to the protein, the electrostatic contribution varies among the ligands by as much as 2 kcal/mol. Moreover, although the ligands are rigid and similar in size, the entropic terms also significantly affect the relative binding affinities (by up to 2.7 kcal/mol). The present approach to solvation and entropy may allow the ranking of the ligands to be considerably improved at a cost that makes the method applicable to the optimization of lead compounds or to the screening of small collections of ligands.