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

Members of the parvalbumin (PV) family of calcium (Ca2+) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca2+ affinities and selectivities against competing ions like Mg2+ can widely vary. We conducted molecular dynamics simulations of several alpha-parvalbumin (alpha PV) constructs with micromolar to nanomolar Ca2+ affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca2+ affinity (approximate to-18 kcal/mol) relative to the wild type (WT) (approximate to-22 kcal/mol) and an S55D/E59D variant 4 with enhanced affinity (approximate to-24 kcal/mol). Additionally, we also examined the binding of Mg2+ to these isoforms, which is much weaker than Ca2+. 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 Mg2+ from Ca2+, they did not indicate that Ca2+ 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 alpha PV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E alpha PV. Meanwhile, Mg-2+ binding was favored for the S55D/E59D alpha PV and D94S/G98E alpha PV 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 alpha-helicity, beta-sheet character, and hydrogen bonding. Hence, Ca2+ affinity and selectivity against Mg2+ 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 alpha PV ion binding that are likely shared by members of the broad family of CBPs.

Automated summary

The study compared the binding affinities of different alpha-parvalbumin constructs from the PV family to Ca2+ and Mg2+, revealing that structural features beyond oxygen coordination in the binding loop significantly influence ion binding.