Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1-CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24-26 and 32′34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1-CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more "open" conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly. © 2010 American Chemical Society.