Exploring calcium ion-dependent effect on the intermolecular interaction between human secreted phospholipase A2 and...

Human secreted phospholipase A₂ (hsPLA₂) is a small calcium ion (Ca²⁺)-regulatory protein secreting from platelets, eosinophils and T-lymphocytes, which has been established as an important biomarker and potential target for the diagnosis and therapy of coronary artery disease. Short peptide inhibitors are used to competitively suppress the enzymatic activity of hsPLA₂. Here, Ca²⁺ effect on the intermolecular recognition and interaction between hsPLA₂ and its peptide inhibitors is investigated systematically by using molecular modeling and bioinformatics analysis. Dynamics simulations reveal that the hsPLA₂ structure bound with Ca²⁺ is rather stable and has low thermal motion; removal of Ca²⁺ considerably increases structural flexibility and intrinsic disorder of the protein. Energetics calculations suggest that presence of Ca²⁺ can effectively promote the interaction of hsPLA₂ with peptide inhibitors. In particular, the local substructures of hsPLA₂ such as helix H1, loop L2 and double-stranded β-sheet DS that participate in peptide recognition are involved in or nearby Ca²⁺-coordinating site and can be directly stabilized by the Ca²⁺. In addition, a significant concentration-dependent effect of Ca²⁺ on peptide-hsPLA₂ binding is observed in vitro, that is, a little of Ca²⁺ can largely improve peptide binding affinity, but high Ca²⁺ concentration does not increase the affinity substantially. The correlation between calculated free energy and experimental binding affinity over different peptide inhibitors is improved considerably by adding Ca²⁺ to hsPLA₂. Specifically, the FLSYK peptide can generally bind to Ca²⁺-bound hsPLA₂ with a moderate or high affinity (K_d ranges between 56 and 210 μM), but have only a modest affinity or even nonbinding to Ca²⁺-free hsPLA₂ (K_d > 400 μM or = n.d.).