Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.304063v1?rss=1 Authors: Immadisetty, K., Sun, B., Kekenes-Huskey, P. Abstract: Parvalbumin (PV) is a calcium binding protein expressed in humans, fish and avian species. In these organisms, the calcium (Ca2+) affinities of specific PV isoforms can vary by orders of magnitude. Despite the availability of high resolution structural data for many PV isoforms, the structural bases for how such proteins confer widely-varying divalent Ca2+ affinities and selectivities against common ions like magnesium (Mg2+) has been difficult to rationalize. We therefore conducted molecular simulations of several -pavalbumin (-parvalbumin (PV)) constructs with Ca2+ affinities in the micromolar to nanomolar ranges to identify properties of conformations that contribute to their wide-ranging binding constants and selectivities against Mg2+. Specifically we examined D94S/G98E construct with a reported lower Ca2+ affinity ({approx} -18.2 kcal/mol) relative to the WT ({approx}-22 kcal/mol), an S55D/E59D variant with enhanced affinity ({approx}-24 kcal/mol), and a truncated variant of PV with weak affinity ({approx}-12.6 kcal/mol). We performed molecular dynamics simulations of these constructs and assessed their Ca2+ and Mg2+ binding properties using scores from molecular mechanics generalized Born approximation (MM/GBSA), ion/oxygen coordination patterns and thermodynamics via mean spherical approximation (MSA) theory, as well as via metrics of protein structure and hydration. Our key findings are that although MM/GBSA and MSA scores successfully rank-ordered the variants according to their previously-published affinities and Mg2+ selectivity, importantly, properties of Ca2+ loops in CBPs such as coordination, and charge are alone insufficient to rationalize their binding properties. Rather, Ca2+ affinity and selectivity against 2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions from protein folding and solubility. Our findings broaden our understanding of the molecular bases governing PV ion binding that are likely shared by many Ca2+ binding proteins. Copy rights belong to original authors. Visit the link for more info