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Backbone torsional strain has been implicated as a cause of rate enhancement in a class of autoprocessing proteins performing proteolysis and protein splicing. In the autoproteolytic protein domain SEA, folding and proteolytic activity have experimentally been shown to be coupled with about 7 kcal/mol of folding free energy available for catalysis. Here, we have examined the catalytic strategy of SEA with molecular dynamics simulations, potential of mean force free energy profiles, and B3LYP/6-311G(d,p) density functional calculations. A quantitative estimate of the free energy stored as protein strain (about 8 kcal/mol), that is available for catalyzing the cleavage reaction, is obtained and found to be in excellent agreement with thermodynamic and kinetic data. It is further shown that there is strong coupling between folding and reaction coordinates leading to reactant state destabilization in the direction of folding and transition state stabilization along the reaction coordinate. This situation is different from the preorganized active site model in that the fully folded transition state stabilizing structure is not realized until the reaction barrier is surmounted.

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

The linear interaction energy method (LIE), which combines force field based molecular dynamics (MD) simulations and linear response theory, has previously been shown to give fast and reliable estimates of ligand binding free energies, suggesting that this type of technique could be used also in a high-throughput fashion. However, a limiting step in such applications is the assignment of atomic charges for compounds that have not been parametrized within the given force field, in this case OPLS-AA. In order to reach an automatable solution to this problem, we have examined the performance of nine different ab initio and semiempirical charge methods, together with estimates of solvent induced polarization. A test set of ten HIV-1 reverse transcriptase inhibitors was selected, and LIE estimates of their relative binding free energies were calculated using the resulting 23 different charge variants. Over 800 ns of MD simulation show that the LIE method provides excellent estimates with several different charge methods and that the semiempirically derived CM1A charges, in particular, emerge as a fast and reliable alternative for fully automated LIE based virtual screens with the OPLS-AA force field. Our conclusions regarding different charge models are also expected to be valid for other types of force field based binding free energy calculations, such as free energy perturbation and thermodynamic integration simulations.

Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --O or N --S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.