New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.

All mammalian cells express three closely related Ras proteins: H-Ras, K-Ras and N-Ras that promote oncogenesis when mutationally activated at codons 12, 13 or 61. Despite a high degree of similarity between the isoforms, K-Ras mutations are far more frequently observed in cancer and each isoform displays preferential coupling to particular cancer types. We have examined the mutation spectra of Ras isoforms curated from large-scale tumour profiling and found that each isoform exhibits surprisingly distinctive codon mutation and amino acid substitution biases. These were unexpected given that these mutations occur in regions that share 100% amino acid sequence identity between the three isoforms. Importantly, many of the mutational biases were not due to differences in exposure to mutagens because the patterns were still evident when compared within specific cancer types. We discuss potential genetic and epigenetic mechanisms together with isoform-specific differences in protein structure and signalling that may promote these distinct mutation patterns and differential coupling to specific cancers.

FTMAP is available as a server at http://ftmap.bu.edu/.

Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the γ-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the γ-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca 2þ and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.he Ras/Raf/MEK/ERK signaling pathway is the most well studied of five known mitogen activated protein kinase (MAPK) cascades involved in the mediation and timing of signaling events in the cell (1). This pathway is activated by Ras GTPase in response to extracellular signals and is involved in the control of cell proliferation, differentiation, and survival (2). In its resting state Ras is bound to GDP and is in a conformation in which it does not interact with Raf or other effector proteins (3). Guanine nucleotide exchange factors facilitate the release of GDP (4). Once the more abundant GTP binds, the Ras switch I (residues 30-40) and switch II (residues 60-76) regions become poised for interaction with effector proteins, leading to the propagation of signal transduction cascades. Ras has a low intrinsic rate of GTPase activity that is enhanced by 3-5 orders of magnitude in the presence of GTPase activating proteins (GAPs), resulting in depletion of Ras-GTP as the switch is turned off (5).The biochemical properties of Ras and its oncogenic mutants have been well characterized in the absence of Raf or other factors (6, 7), and numerous structures of wild-type and oncogenic Ras mutants have been used to study the possible mechanisms through which Ras becomes defective in its ability to hydrolyze GTP (8-12). The switch regions in these structures, solved from the crystal form with symmetry of space group P3 2 21, are modulated by crystal contacts to resemble the switch I and switch II conformations found in the Ras/RasGAP complex (5, 13). Since the structure of this complex has elucidated the m...

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

This review article begins with a discussion of fundamental differences between substrates and inhibitors, and some of the assumptions and goals underlying the design of a new ligand to a target protein. An overview is given of the methods currently used to locate and characterize ligand binding sites on protein surfaces, with focus on a novel approach: multiple solvent crystal structures (MSCS). In this method, the X-ray crystal structure of the target protein is solved in a variety of organic solvents. Each type of solvent molecule serves as a probe for complementary binding sites on the protein. The probe distribution on the protein surface allows the location of binding sites and the characterization of the potential ligand interactions within these sites. General aspects of the application of the MSCS method to porcine pancreatic elastase is discussed, and comparison of the results with those from X-ray crystal structures of elastase/inhibitor complexes is used to illustrate the potential of the method in aiding the process of rational drug design.

Porcine pancreatic elastase has been used as the model enzyme in the design and development of a crystallographic method that allows mapping of the binding surface of a protein by solving its crystal structure in a variety of organic solvents. The ultimate goal of this method is to aid in the process of drug design, where each of the chosen organic molecules represents a given functional group in a larger inhibitor molecule. This method of multiple solvent crystal structures (MSCS) has a theoretical counterpart in the method of multiply copy simultaneous search (MCSS) (Miranker, A.; Karplus, M. Proteins: Struct., Funct., Genet. 1991, 11, 29-34) and is the first experimental method that can be used as a check to the theory. The MSCS method is presented here with acetonitrile as the probe organic solvent. The procedure involved does not cause significant changes in the structure of elastase as compared to the structure in aqueous solution, and the positions found for the acetonitrile molecules in the active site are compared to those of similar functional groups belonging to known inhibitors bound to elastase.

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.