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

Abstract

Members of the parvalbumin (PV) family of calcium (Ca 2+ ) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca 2+ affinities and selectivities against competing ions like Mg 2+ can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca 2+ 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 2+ 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 2+ to these isoforms, which is much weaker than Ca 2+ . 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 2+ from Ca 2+ , they did not indicate that Ca 2+ 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 2+ binding by −20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg 2+ 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 2+ affinity and selectivity against Mg 2+ 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.

Importance of AB domain in parvalbumins’ calcium binding affinity

“…Note that C-terminus of the D-helix interacts with the AB domain. Contrary to our earlier study [30], we find that the number of coordinating oxygens in the EF hand binding sites' are sufficient to completely rationalize observed changes in affinity.…”

“…Therefore, we rather show that reorganization energy of forming the holo state upon ion binding relative to the apo rank-ordered the variants correctly. We have successfully demonstrated the usefulness of this strategy in a previous study [30].…”

“…MD simulations [24] have facilitated detailed analyses of Ca 2+ binding in intact proteins. More recent applications include refinement of force field parameters for Ca 2+ [25, 26], Ca 2+ binding energetics in EF-hand containing proteins [27, 28, 29, 30], effects of Ca 2+ binding on protein function [31, 32, 33], mutations that disrupt the binding of Ca 2+ [34, 30] and differential modulation of purinergic receptorsn by Mg 2+ and potassium (K + ) [35].…”

Parvalbumin Role in Epilepsy and Psychiatric Comorbidities: From Mechanism to Intervention

“…A recent study indicates that, although the intrinsic characteristics of the EF-hand contribute strongly to the selectivity of Ca 2+ in α-parvalbumin, allosteric changes affecting secondary and tertiary structures play a significant role in differentiating strong from weak Ca 2+ binding. The authors also report that Ca 2+ affinity and selectivity against Mg 2+ are properties that emerge both from local effects at ion binding sites and non-local contributions elsewhere ( Immadisetty et al, 2021 ). In addition to the morphofunctional characteristics of each parvalbumin isoform, its location is also an essential factor in the myriad of physiological processes regulated by these Ca 2+ /Mg 2+ binding proteins ( Schwaller, 2009 ).…”

Elucidating the molecular basis of spontaneous activation in an engineered mechanosensitive channel

“…MD is a powerful computational tool that is used, among other things, to study various therapeutically important targets at an atomic level [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] . Several MD studies using coarse-grained MD [41] , [42] , [43] or biased MD [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , have been conducted thus far for studying the activation of wild-type (WT) MscL.…”