Molecular Dynamics Studies of Point Mutations of Cardiac Troponin C and Annexin

Abstract

Calcium binding proteins are biologically important for their ability to convert changes in calcium ion concentration to functional changes in proteins. All calcium binding proteins use a calcium sensing motif that changes the protein conformation and dynamics when associated with calcium ions. Dysfunction in calcium binding proteins has been linked to many diseases such as cancer and heart disease. In this study, models of mutated proteins were generated and Molecular Dynamics simulations were used to study Annexin and Cardiac Troponin C (cTnC). Annexin A1 is an important protein that is known to induce membrane aggregation, while cardiac Troponin C (cTnC) regulates cardiac muscle contraction. This study focuses on the impact of mutations on Annexin A1 and cTnC with the objective to gain insight of how these changes are made by mutations. The ultimate goal of this research is to use the knowledge of the effects of point mutations on calcium binding proteins dynamics to treat disease. Multiple MD simulations were performed for the full length wild type Annexin A1, the full length A1 mutations S27E and S27A, as well as the N-terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKGS²⁷KGGPGSAVSPYPTFN) of wild-type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350ns were generated and analyzed to examine the changes of the core domain calcium binding affinity, core domain and N-terminal domain structures, and N-terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium-binding affinity of the core domain of A1. However, the S27A mutation made the N-terminal domain of A1 less helical, and made the N-terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial or have no effect on the membrane aggregation process. On the contrary, the S27E mutation made the N-terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for previous experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild-type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Molecular Dynamics simulations of about 145 ns total were performed for wild type cardiac troponin C and two Site II mutations, D65A and S69C. The simulation trajectories were analyzed using MMPBSA, MMGBSA, RMSF, RMSD, cluster analysis and various visualization programs. The results showed that the mutations caused a decrease in calcium binding affinity that is similar to what was shown in previous studies. The loss of calcium binding affinity can be attributed to a loss of binding at two residues, Ser 69 and Asp 73. Cross-correlation analysis shows that inter-domain interactions change dramatically when cTnC interacts with the other two subunits of cTn. Possible downstream effects of cTnC point mutations were seen through the changes of inter-domain flexibility, orientation and position from that of wild-type. The accessibility of cTnI to the hydrophobic binding pocket located within the N-terminal domain of cTnC was more restricted for the D65A and S69C cTnC mutants than wild-type cTnC. The restriction of cTnI to the switch segment suggests that the mutant cTnC proteins have a negative effect on the downstream function of cTn. The changes observed in this study on Site II point mutations provide insight into disease causing mechanisms of the D65A and S69C mutations.