Molecular dynamics study of Ca2+ binding loop variants of parvalbumin with modifications at the 'gateway' position

The helix-loop-helix (i.e. EF-hand) Ca2+ ion binding motif is characteristic of a large family of high-affinity Ca2+ ion binding proteins, including the parvalbumins and calmodulins, In this paper we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B). In all variants examined, both whole protein and fragments thereof, the ninth loop residue in the Ca2+ binding coordination site in the CD helix-loop-helix region (the so-called 'gateway' residue) has been mutated, The three gateway mutations examined are arginine, which has never been found at the gateway position of any EF-hand protein, cysteine, which is the residue observed least in natural EF-hand sites, and serine, which is the most common (by far) non-acidic residue substitution at this position in EF-hand proteins in general, but never in parvalbumins, Results of the molecular dynamics simulations indicate that all three modifications are disruptive to the integrity of the mutated Ca2+ binding site in the whole parvalbumin protein. In contrast, only the arginine and cysteine mutations are disruptive to the integrity of the mutated Ca2+ binding site in the CD fragment of the parvalbumin protein, Surprisingly, the serine variant of the CD helix-loop-helix fragment exhibited remarkable stability during the entire molecular dynamics simulation, with retention of the Ca2+ binding site. These results indicate that there are no inherent problems (for Ca2+ ion binding) associated with the sequence of the CD helix-loop-helix fragment that precludes the incorporation of serine at the gateway position, Since the CD site is totally disrupted in the whole protein serine variant, this indicates that the Ca2+ ion binding deficiencies are most likely related to the unique interaction that exists between the paired EF-hands in the whole protein. Our theoretical results correlate well with previous studies on engineered EF-hand proteins and with all of our experimental evidence on the silver hake parvalbumin.