Importance of AB domain in parvalbumins’ calcium binding affinity

Members 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.