Structural Changes beyond the EF-Hand Contribute to Apparent Calcium Binding Affinities: Insights from Parvalbumins.

Members of the parvalbumin (PV) family of calcium (Ca) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca affinities and selectivities against competing ions like Mg can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca affinity (≈-18 kcal/mol) relative to the wild type (WT) (≈-22 kcal/mol) and an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol). Additionally, we also examined the binding of Mg to these isoforms, which is much weaker than Ca. We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition. While the MSA scores differentiated Mg from Ca, they did not indicate that Ca binding affinities at the binding loop differed between the PV isoforms. Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg binding was favored for the S55D/E59D αPV and D94S/G98E αPV variants by -18.32 and -1.65 kcal/mol, respectively. These energies implicate significant contributions to ion binding beyond oxygen coordination at the binding loop, which stemmed from changes in α-helicity, β-sheet character, and hydrogen bonding. Hence, Ca affinity and selectivity against Mg are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions elsewhere. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by members of the broad family of CBPs.