MOLECULAR-DYNAMICS STUDY OF CALBINDIN D-9K IN THE APO AND SINGLY AND DOUBLY CALCIUM-LOADED STATES

Molecular dynamics simulations based on detailed atomic models are use d to examine the structure and dynamics of calbindin D-9k, a protein p ossessing a pair of EF-hands able to bind two calcium ions in a cooper ative fashion, Trajectories for the apo and singly tin the C-terminal binding site) and doubly loaded structures are generated and analyzed. Each system is solvated in a 27 Angstrom radius sphere of 2,285 expli cit water molecules. The influence of the remaining bulk is incorporat ed through a stochastic boundary potential including a solvent reactio n held, Long-range electrostatic interactions are treated with a speci al method and are not truncated. The average structural and dynamic pr operties upon calcium binding are studied at the atomic level to gain insight into the cooperative interactions between the two binding site s. Results from the trajectories are compared with data from nuclear m agnet;ic resonance (NMR) spectroscopy and X-ray crystallography. NMR N -15 and C-13(alpha) backbone relaxation order parameters and crystallo graphic B-factors are calculated. Generally, there is a good qualitati ve agreement between calculated and observed properties. Results confi rm that the doubly loaded state is closer, both structurally and dynam ically, to the singly loaded state than either of these is to the apo state. It is observed that both hydrogen bonding and the packing of no npolar side chains contribute to the coupling between the calcium bind ing sites. Two backbone-to-backbone hydrogen bonds linking the calcium -binding EF-hands (Leu23-O ... HN-Val61 and Val61-O ... HN-Leu23) are sensitive to the state of occupancy. Residues Leu23 and Val61 exhibit the smallest rms fluctuations of the entire protein in the D state. In addition, the van der Waals interaction of Val61 with the rest of the protein varies with the calcium-binding state, (C) 1998 Wiley-Liss, I nc.