Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Molecular Dynamics Simulations and Metal Ion Binding Properties of Wild-Type and Mutational Variants of Functional Fragments of Silver Hake Parvalbumin (Isoform B)"

The helix-loop-helix (i.e., EF-hand) Ca2+ ion binding motif is characteristic of a large family of high-affinity calcium ion binding proteins, including the parvalbumins, oncomodulins and calmodulins. In this work we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B) and on functional fragments of this protein, consisting of the first four helical regions (the ABCD fragment), and the internal helix-loop-helix region (the CD fragment). In both whole protein and protein fragments (i.e., ABCD and CD fragments), the 9th loop residue in the calcium ion binding site in the CD helix-loop-helix region (the so-called "gateway" position) has been mutated from glutamic acid to aspartic acid. Aspartic acid is one of the most common residues found at the gateway position in other (non-parvalbumin) EF-hand proteins, but has never been found at the gateway position of any parvalbumin. [Interestingly, aspartic acid does occur at the gateway position in the closely related rat and human oncomodulins.] Consistent with experimental observations, the results of our molecular dynamics simulations show that incorporation of aspartic acid at the gateway position is very disruptive to the structural integrity of the calcium ion coordination site in the whole protein. The aspartic acid mutation is somewhat less disruptive to the calcium ion coordination sites in the two parvalbumin fragments (i.e., the ABCD and CD fragments), presumably due to the higher degree of motional freedom allowable in these protein fragments. One problem associated with the E59D whole protein variant is a prohibitively close approach of the aspartate carboxyl group to the CD calcium ion observed in the energy-minimized (pre-molecular dynamics) structure. This steric situation does not emerge during energy-minimization of the wild-type protein. A figure showing the superpositioning of the calcium ion ligands in the CD site for the wild-type (gray color) and E59D (CPK colors) whole protein variants is presented below. The close approach of the aspartate 59 (orange) carboxyl group to the CD calcium ion (yellow for the E59D variant) is clearly revealed in this image. Note that the CPK images representing the calcium ions are at 50% of their normal size.

The damage to the structural integrity of the calcium ion coordination site in the whole protein E59D variant is not relieved during the molecular dynamics simulation. In fact, during the course of the 300 picosecond simulation, all of the calcium ion ligands leave the primary coordination sphere. In addition, the conserved hydrogen-bonds (in the short b-sheet structure) that links the CD site to the symmetry-related EF site (in the non-mutated whole protein) is also somewhat disrupted in the E59D

Kelly M. Elkins, Kamau Fahie, Rebecca Pitts, Sandra P. Revett and Donald J. Nelson

Department of Chemistry and Biochemistry, Clark University Worcester, MA 01610 D.J. Nelson : Phone - 508-793-7121 Fax - 508-793-8861 Email - dnelson@clarku.edu