The nature of the ion binding interactions in EF-hand peptide analogs: free energy simulation of Asp to Asn mutations

The binding of the La3+ ion to a tridecapeptide, which is a model for the EF-hand in calcium-binding proteins, is studied hi solution by free energy simulations. The calculations analyze the effect on the La3+ ion binding of the mutation of Asp to Asn for side chains that interact directly with the ion. The results are compared with the measurements of Marsden.B.J., Hodges, R.S. and Sykes, B.D. (1989) Biochemistry, 28,8839, on the same system. They found that the Asp to Asn mutation has only a small effect on the binding; the observed differences in the free energies on changing one Asp to an Asn are between -0.3 and 1.8 kcal/ mol. This result is analyzed by alchemical simulations for the tridecapeptide in the bound Qoop) structure and free (extended) form. The free energy changes due to the mutation of an Asp to an Asn are large and positive for both the bound and free forms. However, since the values of the free energy changes are calculated to be similar hi the two forms, the difference in the binding free energy of Asp and Asn peptides is found to be small, in agreement with experiment. By use of thermodynamic integration, the various contributions to the free energy changes are estimated. In the com-plexed form, the Asp to Asn mutation is favored by the reduction in the repulsive interaction with other charged residues of the peptide; it is disfavored by the reduction of the stabilization of the ion and the surrounding water has a small effect. When the peptide adopts an extended conformation in the absence of the ion, the mutation Asp to Asn is strongly disfavored by the interactions with the water and is favored by the interactions within the peptide. The results demonstrate the essential role of contributions to the binding of EF-hands from interactions other than those between the ion and the charged amino acid side chains. The results obtained from the simulations suggest, in accord with crystal structures of La3+ bound to various ligands, that the calcium-binding loop complexed with La3+ in solution has a significantly different structure from that observed hi proteins.