Entropic contributions and the influence of the hydrophobic environment in promiscuous protein-protein association

Abstract

The mechanisms by which a promiscuous protein can strongly interact with several different proteins using the same binding interface are not completely understood. An example is protein kinase A (PKA), which uses a single face on its docking/dimerization domain to interact with multiple A-kinase anchoring proteins (AKAP) that localize it to different parts of the cell. In the current study, the configurational entropy contributions to the binding between the AKAP protein HT31 with the D/D domain of RII alpha-regulatory subunit of PKA were examined. The results show that the majority of configurational entropy loss for the interaction was due to decreased fluctuations within rotamer states of the side chains. The result is in contrast to the widely held approximation that the decrease in the number of rotamer states available to the side chains forms the major component. Further analysis showed that there was a direct linear relationship between total configurational entropy and the number of favorable, alternative contacts available within hydrophobic environments. The hydrophobic binding pocket of the D/D domain provides alternative contact points for the side chains of AKAP peptides that allow them to adopt different binding conformations. The increase in binding conformations provides an increase in binding entropy and hence binding affinity. We infer that a general strategy for a promiscuous protein is to provide alternative contact points at its interface to increase binding affinity while the plasticity required for binding to multiple partners is retained. Implications are discussed for understanding and treating diseases in which promiscuous protein interactions are used.

Fig. 1.

Occupancy (%) of backbone dihedral angles. (a) Asp-1ψ of free Ht31pep. (b) Asp-1ψ of bound Ht31pep. (c) Pro-7ψ of the free D/D protomer II. (d) Pro-7ψ of the bound D/D protomer II.

Fig. 2.

Flipping of an amide group. (a) Pro-7ψ of the free D/D protomer II. (b) Pro-7ψ of the bound D/D protomer II. (c) Gly-8 φ of the free D/D protomer II. (d) Gly-8 φ of the bound D/D protomer II.

Fig. 3.

Distinct side-chain conformations found during the MD simulations. (Left) Free Ht31pep. (Right) Bound Ht31pep. x axis, sample saved during the simulations at time x; y axis, population for each distinct conformation (%). The x axis combines three independent simulations. These samples were taken every 1 ps based on the time evolution during the MD simulation. If a sample has 0 population (y value = 0) at sample x, then it means that the snapshot saved at a certain time is a repeat.

Fig. 4.

Number of rotameric states and their transition frequency (%) observed during the simulations. Open squares, side-chain dihedrals of free Ht31pep; filled circles, side-chain dihedrals of bound Ht31pep; x axis, dihedral index; left y axis, transition frequency; right y axis, number of rotameric states. Dihedrals of residues that interact with the D/D domain are indicated by a cross aligned at the top of the plot. Note that the hydrophobic environment may allow a side-chain dihedral to have more rotameric states in its bound state (see dihedrals 2, 16, and 20) or to have more frequent transitions between different states, e.g., dihedral 3.

Fig. 5.

Relationship between alternative contacts and configurational entropy. (a) Plot of the total number of mutually exclusive alternative contacts for atoms that participate in hydrophobic contacts versus their associated configurational entropy. The correlation coefficient for the linear fit of the averages was 0.95, with a P value of 0. The correlation was a property of the average entropy at each alternative contact. The result indicates that the configurational entropy of a hydrophobic atom is, on average, directly proportional to the number of alternative hydrophobic contacts available to that atom. (b) Plot of the total number of alternative hydrophobic contacts of hydrophobic atoms in the Ht31 peptide that were gained upon complex formation versus the average configurational entropy change associated with these atoms upon binding. The correlation coefficient for the linear fit of the averages was 0.88, with a P value of 0.0008. The result demonstrates that when there is a gain in the number of available alternative contacts available to an atom upon complex formation, there will be higher configurational entropy associated with that atom.