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

Abstract

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on cell surface signals focal adhesion disassembly. Residues of Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT are critical for TSP1-CRT binding (Faseb J, 22:3968, 2008; J Biol Chem, 268:26784, 1993). This study investigated molecular and structural basis for the effect of these key residues in TSP1 and CRT on TSP1-CRT binding. Based on a validated TSP1-CRT complex structure (Biochemistry, 47:3685, 2010), we adopted steered molecular dynamics simulations to determine the effect of the mutations of these key residues on TSP1-CRT binding and validated the simulation results with experimental observation. We further performed 30 ns molecular dynamics simulations for wild type TSP1, CRT, TSP1 K24A&K32A mutant and mutant CRT (residues 24-26 & 32-34 mutated to Ala) and studied the conformational and structural changes of TSP1 and CRT by the mutations of the critical residues. Results showed that mutations of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 in CRT to Ala result in a shortened β strand in the binding site, decreased hydrogen bond occupancy for β strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamic correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motions between the residues in protein. These changes could directly contribute to the loss or reduced binding of TSP1-CRT complex and further affect TSP1-CRT binding induced cellular activities. Results from this study provide molecular and structural insight for the role of the critical residues in TSP1 and CRT in TSP1-CRT binding.