A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum͞classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.enzyme catalysis ͉ molecular dynamics D ihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate using nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme (1). This reduction is an essential step for the biosynthesis of purines, pyrimidines, and amino acids. DHFR has been fostered as a pharmacological target for anticancer drugs and antibacterial agents because of its physiological importance for normal folate metabolism (2). As a result, DHFR has been studied extensively with a wide range of experimental and theoretical approaches. Fig. 1 depicts the 3D structure of DHFR and identifies important loop regions, such as the Met-20 loop (residues 9-24), the ␤F-␤G loop (residues 116-132), and the ␤G-␤H loop (residues 142-150), as well as the adenosine binding domain (residues 38-88).The detailed mechanism for DHFR has been determined from kinetic studies of the Escherichia coli species (1, 3). X-ray crystallographic structures of DHFR in binary and ternary complexes (4) indicate that the enzyme assumes different conformations along the reaction pathway. Furthermore, NMR relaxation experiments (5-7) imply that the binding of the substrate and the coenzyme induces conformational changes of structural elements both in and distal to the active site, including the Met-20 and ␤F-␤G loops. Classical molecular dynamics simulations of the reactant ternary complex with DHF identified correlated and anticorrelated motions involving many of the same spatial regions as implicated by the dynamic NMR measurements (8). These correlations are absent in the product complex with 5,6,7,8-tetrahydrofolate. Mutant DHFR enzymes with reduced activity exhibit a reduction in these correlated motions compared with the wild-type (WT) system (9, 10).Hybrid quantum͞classical molecular dynamics simulations provided evidence of a network of coupled motions ext...

Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-beta-lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye-Hückel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 A, or rotation angles of >60 degrees from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein-protein docking is discussed.

Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by ⌽ analysis of the encounter complex and transition state of the RalGDS M26K,D47K,E54K ͞Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

Previous studies of E. coli dihydrofolate reductase (ecDHFR) have demonstrated that residue G121, which is 19Å away from the catalytic center, is involved in catalysis and long distance dynamical motions were implied. Specifically, the ecDHFR mutant G121V has been extensively studied by various experimental and theoretical tools, and the mutation's effect on kinetic, structural, and dynamical features of the enzyme explored. The current work examined the effect of this mutation on the physical nature of the catalyzed hydride transfer step by means of intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters as described before for the wild type ecDHFR (Sikorski et al. 2004, J. Am. Chem. Soc., 126, 4778-4779). The temperature dependence of initial velocities was used to estimate activation parameters. Isotope effects on the preexponential Arrhenius factors, and the activation energy could be rationalized by an environmentally coupled hydrogen tunneling model, similar to the one used for the wild type enzyme. Yet, in contrast to the wild type, fluctuations of the donor-acceptor distance were now required. Secondary (2°) KIEs were also measured for both H and D transfer and, as in the case of the wild type enzyme, no coupled motion was detected. Despite these similarities, the reduced rates, the slightly inflated 1° KIEs and their temperature dependence, together with relatively deflated 2° KIEs, indicate that the potential surface prearrangement was not as ideal as for the wild type enzyme. These findings support theoretical studies suggesting that the G121V mutation lead to a different conformational ensemble of reactive states and less effective rearrangement of the potential surface, but has only small effect on H-tunneling.Dihydrofolate reductase from Escherichia coli (ecDHFR) is a small monomeric enzyme (18 kDa) that consists of eight-stranded β-sheet and four α-helices connected with several loop regions. It catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7, with the concomitant oxidation of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) to NADP + , in which a hydride is stereospecifically transferred from the pro-R C4 position of the nicotinamide ring to the si face of the C6 position of pterin. This enzyme helps maintain intracellular pools of THF used in the biosynthesis of purine nucleotides and some amino acids. Furthermore the essential role of DHFR in DNA synthesis and in a variety of anabolic pathways makes it a common target for antiproliferative therapeutics. Due to its biological and pharmacological importance, and it being a small monomeric enzyme, DHFR has been the subject of intensive structural and kinetic investigation over many years, serving as a paradigm of enzymatic systems in many experimental and theoretical studies (1)(2)(3)(4)(5)(6)(7)(8). † This work was supported by NIH R01 GM65368-01 and NSF CHE-0133117 to A.K. The complete kinetic scheme for wild type ecDHFR was derived from equilibrium binding, steady-sta...