Resurrecting prehistoric parvalbumins to explore the evolution of thermal compensation in extant Antarctic fish parvalbumins

Sun²,

Kekenes‐Huskey³

2021

J. Phys. Chem. B

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.

Section: Discussionsupporting

confidence: 74%

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

Sun²,

Kekenes‐Huskey³

2021

J. Phys. Chem. B

“…From this perspective, the presence of the AB domains may amortize the thermodynamic cost of folding the protein upon binding Ca 2+ , which would otherwise reduce the αPV apparent Ca 2+ affinity. In support of this interpretation, lysine-to-asparagine mutations of the AB domain sites A8 and H26 in PV constructs modeled after Antartic fish were found to increase Ca 2+ binding affinity and was attributed to forming additional hydrogen bonds within the AB or between AB and CDEF domains [63]. Overall, our observations highlight the importance of AB/CDEF domain interactions for maintaining high Ca 2+ binding affinity.…”

Section: The Displacement Of Loosely-bound Waters In the CD And Ef Losupporting

confidence: 74%

Structural determinants of calcium binding beyond the EF-hand binding site: a study of alpha parvalbumins

Sun²,

Kekenes‐Huskey³

2020

Preprint

1AbstractParvalbumin (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 α-pavalbumin (α-parvalbumin (αPV)) 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 a D94S/G98E construct with a reported lower Ca2+ affinity (≈ −18.2 kcal/mol) relative to the WT (≈ −22 kcal/mol), an S55D/E59D variant with enhanced affinity (≈ −24 kcal/mol), and a truncated variant of αPV with weak affinity (≈ −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 Mg2+ 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 αPV ion binding that are likely shared by many Ca2+ binding proteins.

“…From this perspective, the presence of the AB domains may amortize the thermodynamic cost of folding the protein upon binding Ca 2+ , which would otherwise reduce the αPV apparent Ca 2+ affinity. In support of this interpretation, lysine-to-asparagine mutations of the AB domain sites A8 and H26 in PV constructs modeled after Antarctic fish were found to increase Ca 2+ binding affinity and were attributed to forming additional hydrogen bonds within the AB or between AB and CDEF domains [61]. Overall, our observations highlight the importance of AB/CDEF domain interactions for maintaining high Ca 2+ binding affinity.…”

Section: Structural Contributions Beyond Ef-hand Loop In Determining ...supporting

confidence: 74%

Importance of AB domain in parvalbumins’ calcium binding affinity

Jacob-Dolan²

2022

Preprint

1AbstractMembers of the parvalbumin (PV) family of calcium binding proteins are found in a variety of vertebrates, where can they influence neural functions, muscle contraction and immune responses. It was reported that the α-parvalbumin (αPV)s AB domain comprising two α-helices, dramatically increases the proteins calcium (Ca2+) affinity by ≈10 kcal/mol. To understand the structural basis of this effect, we conducted all-atom molecular dynamics (MD) simulations of WT αPV and truncated α-parvalbumin (ΔαPV) constructs. Additionally, we also examined the binding of magnesium (Mg2+) to these isoforms, which is much weaker than Ca2+ (Mg2+ actually does not bind to the ΔαPV). Our key finding is that ‘reorganization energies (RE)’ assessed using molecular mechanics generalized Born approximation (MM/GBSA) correctly rank-order the variants according to their published Ca2+ and Mg2+ affinities. The of the ΔαPV compared to the wild-type (WT) is 415.57±0.55 kcal/mol, indicating that forming a holo state of ΔαPV in the presence of Ca2+ incurs a greater reorganization penalty than the WT. This is consistent with the ΔαPV exhibiting lesser Ca2+ affinity than the WT (≈9.5 kcal/mol). Similar trend was observed for Mg2+ bound variants as well. Further, we screened for metrics such as oxygen coordination of EF hand residues with ions and found that the total oxygen coordination number (16 vs. 12 in WT:Ca2+ and ΔαPV:Ca2+) correlate with the reported ion affinities (−22 vs. −12.6 kcal/mol in WT:Ca2+ and ΔαPV:Ca2+), which indicates that AB domain is required for the protein to coordinate with maximal efficiency with the binding ions. To our surprise, no significant differences were observed between the Mg2+ bound WT and ΔαPV isoforms. Additionally, we have screened for factors such as total number of waters, hydrogen bonds, protein helicity and β-content for the entire protein, which enables us to understand the impact of lack of AB domain on the entire structure and not just binding sites. Our data indicate that AB improves the overall helicity (≈5%) in apo as well as holo forms. Particularly, AB increases α-helicity in the D-helix residues (i.e., 60–65) upon ion binding by ≈35% (90% vs. 55% in the Ca2+ bound WT and ΔαPV, 60% vs. 20% in the Mg2+ bound WT and ΔαPV), which likely contributes to high Ca2+ binding affinity. On the contrary, no significant effect on the overall β-content was observed. Similarly, increased dehydration (≈50) and increase in total number of hydrogen bonds (≈7) were observed upon ion binding in both the WT and ΔαPV systems, however, no significant differences were observed between the WT and ΔαPV variants and also between Ca2+ and Mg2+ isoforms. We speculate that this is due to the partially folded apo state that was captured in our MD simulations, which might not be physiologically relevant as suggested by NMR experiments [1]. Also, we have identified seven different interactions that might play a key role in binding the AB domain with the CDEF helices, particularly the D22(AB)–S78(CDEF) hydrogen bond. Overall, this study indicates that local (i.e., the EF hands) as well as global factors play a role in improved ion binding due to AB domain.