Structural insight into the role of thrombospondin-1 binding to calreticulin in calreticulin-induced focal adhesion disassembly

Abstract

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.

Figure 1

Protocol for TSP1–CRT complex prediction.

Figure 2

(A) RMSD for the TSP1 N-domain in TSP1–CRT complexes over the 30 ns MD simulation. (B) RMSD for the CRT N-domain and the partial C-domain in TSP1–CRT complexes over the 30 ns MD simulation.

Figure 3

Binding free energy over the 30 ns MD simulations for the constructed TSP1–CRT complex and the mutant complex.

Figure 4

(A) RMSF comparison of CRT N- and P-domains for CRT alone and CRT in the TSP1–CRT complex. (B) Dynamical cross-correlation maps for the degree of correlated motion of the residues in CRT alone (bottom right) and CRT in the TSP1–CRT complex (top left). (C) Distance matrix for CRT alone (bottom right) and CRT in the TSP1–CRT complex (top left).

Figure 5

(A) Definition of the angle for the CRT N-domain with respect to the CRT P-domain. The vector formed by residues 129 and 145 in the CRT N-domain relative to the plane formed by residues 190, 210, and 240 in the CRT P-domain. (B) Angle between the CRT N- and P-domains for CRT alone and CRT in the TSP1–CRT complex.

Figure 6

RMSD of backbone Cα atoms and energy of CRT in ANM–MD simulations. (A) Single CRT. (B) TSP1–CRT complex. The black (minus) and red (plus) curves represent two equally possible directions.

Figure 7

Supposition of the final structure of CRT alone (red) and the structure of the TSP1–CRT complex (blue) after ANM restrained MD simulations.