Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK

Abstract

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.

Thermodynamics and Kinetics of Hsp70 Association with A + U-rich mRNA-destabilizing Sequences

“…6). Second, the binding affinities of RNA substrates for Hsp70 (K d ϭ 28 nM for TNF␣ ARE) are comparable or better than those calculated for binding of peptide substrates to Hsp70 (K d Ն 60 nM) (57), despite the lack of positive charges on the RNA molecules. Taken together, these observations admit the possibility that a site on Hsp70 distinct from the peptide-binding domain may be responsible for contacting RNA substrates.…”

“…First, based on the crystal structure of the Hsp70 peptide-binding domain, the peptide-binding pocket is flanked by acidic amino acid residues, suggesting that cationic substrates would be preferred at this site (52). Peptide substrates flanked by acidic amino acid residues show significantly poorer binding affinity than those flanked by basic residues (57). By contrast, RNA substrates are inherently anionic in solution, and electrostatic interactions play only a minor role in the stability of the Hsp70⅐TNF␣ ARE complex (Fig.…”

Electrostatic Interactions between Peptides and the Molecular Chaperone DnaK

“…Application of these models is, however, limited by computational requirements and the need for precise structural input for all interacting components, including information concerning conformational changes associated with the binding process. In the absence of complete and accurate input information, free-energy calculations often require arbitrary scaling procedures (Novotny et al, 1997;Kasper et al, 2000) that limit the validity of computed results to relative comparisons, but do not always suffice for determinations of absolute thermodynamic quantities of interest.…”

Preferential Substrate Binding Orientation by the Molecular Chaperone HscA

“…This suggests that for this peptide complex of DnaK the electrostatic potential plays a lesser role in determining binding polarity. Studies on the effects of ionic strength on binding of the NRLLLTG peptide to DnaK have indicated an electrostatic free energy of binding about Ϫ1 kcal/mol (46), but empirical calculations based on binding of several charge derivatives to DnaK suggest that nonpolar interactions are the predominant contributor to the free energy of binding (47).…”