Molecular and Structural Insight into the Role of Key Residues of Thrombospondin-1 and Calreticulin in Thrombospondin-1−Calreticulin Binding

摘要

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface signals focal adhesionndisassembly, leading to the intermediate adhesive phenotype, cell migration, anoikis resistance, and collagennstimulation. Residues Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT have been shownnthrough biochemical and cell-based approaches to be critical for TSP1-CRT binding and signaling. Thisnstudy investigated the molecular and structural basis for these key TSP1 and CRT residues in TSP1-CRTnbinding. On the basis of a validated TSP1-CRT complex structure, we adopted steered molecular dynamicsnsimulations to determine the effect ofmutation of these key residues on TSP1-CRT binding and validated thensimulation results with experimental observations. We further performed 30 ns molecular dynamicsnsimulations for wild-type TSP1, CRT, K24A/K32A mutant TSP1, and mutant CRT (residues 24-26 andn32-34mutated to Ala) and studied the conformational and structural changes in TSP1 and CRT as the resultnof mutation of these critical residues. Results showed that mutation of residues 24 and 32 to Ala in TSP1 andnof amino acids 24-26 and 32-34 to Ala in CRT results in a shortened β-strand in the binding site, decreasednhydrogen bond occupancy for β-strand pairs that are located within or near the binding site, increasednconformational flexibility of the binding site, a changed degree of dynamically correlated motion between thenresidues in the binding site and the other residues in protein, and a changed degree of overall correlatedmotionnbetween the residues in the protein. These changes could directly contribute to the loss or weakened bindingnbetween TSP1 and CRT and the resultant effects on TSP1-CRT binding-induced cellular activities. Resultsnfrom this study provide a molecular and structural insight into the role of these critical residues of TSP1 andnCRT in TSP1-CRT binding.