In termination of protein synthesis, the bacterial release factors RF1 and RF2 bind to the ribosome through specific recognition of messenger RNA stop codons and trigger hydrolysis of the bond between the nascent polypeptide and the transfer RNA at the peptidyl-tRNA site, thereby releasing the newly synthesized protein. The release factors are highly specific for a U in the first stop-codon position and recognize different combinations of purines in the second and third positions, with RF1 reading UAA and UAG and RF2 reading UAA and UGA. With recently determined crystal structures of termination complexes, it has become possible to decipher the energetics of stop-codon reading by computational analysis and to clarify the origin of the high release-factor binding accuracy. Here we report molecular dynamics free-energy calculations on different cognate and non-cognate termination complexes. The simulations quantitatively explain the basic principles of decoding in all three codon positions and reveal the key elements responsible for specificity of the release factors. The overall reading mechanism involves hitherto unidentified interactions and recognition switches that cannot be described in terms of a tripeptide anticodon model. Further simulations of complexes with tRNA(Trp), the tRNA recognizing the triplet codon for Trp, explain the observation of a 'leaky' stop codon and highlight the fundamentally different third position reading by RF2, which leads to a high stop-codon specificity with strong discrimination against the Trp codon. The simulations clearly illustrate the versatility of codon reading by protein, which goes far beyond tRNA mimicry.

The binding of blockers to the human voltage-gated Kv1.5 potassium ion channel is investigated using a three-step procedure consisting of homology modeling, automated docking, and binding free energy calculations from molecular dynamics simulations, in combination with the linear interaction energy method. A reliable homology model of Kv1.5 is constructed using the recently published crystal structure of the Kv1.2 channel as a template. This model is expected to be significantly more accurate than earlier ones based on less similar templates. Using the three-dimensional homology model, a series of blockers with known affinities are docked into the cavity of the ion channel and their free energies of binding are calculated. The predicted binding free energies are in very good agreement with experimental data and the binding is predicted to be mainly achieved through nonpolar interactions, whereas the relatively small differences in the polar contribution determine the specificity. Apart from confirming the importance of residues V505, I508, V512, and V516 for ligand binding in the cavity, the results also show that A509 and P513 contribute significantly to the nonpolar binding interactions. Furthermore, we find that pharmacophore models based only on optimized free ligand conformations may not necessarily capture the geometric features of ligands bound to the channel cavity. The calculations herein give a detailed structural and energetic picture of blocker binding to Kv1.5 and this model should thus be useful for further ligand design efforts.

The linear interaction energy (LIE) method in combination with two different continuum solvent models has been applied to calculate protein-ligand binding free energies for a set of inhibitors against the malarial aspartic protease plasmepsin II. Ligand-water interaction energies are calculated from both Poisson-Boltzmann (PB) and Generalized Born (GB) continuum models using snapshots from explicit solvent simulations of the ligand and protein-ligand complex. These are compared to explicit solvent calculations, and we find close agreement between the explicit water and PB solvation models. The GB model overestimates the change in solvation energy, and this is caused by consistent underestimation of the effective Born radii in the protein-ligand complex. The explicit solvent LIE calculations and LIE-PB, with our standard parametrization, reproduce absolute experimental binding free energies with an average unsigned error of 0.5 and 0.7 kcal/mol, respectively. The LIE-GB method, however, requires a constant offset to approach the same level of accuracy.

Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif (Q185 in human eRF1 numbering) is critical for catalysis of the termination reaction on the ribosome. Furthermore, it has been established that Q185 is Nepsilon methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is no such structural effect on the free release factor caused by the Nepsilon methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.