Crystal structure of human chondroadherin: solving a difficult molecular‐replacement problem using de novo de novo models

摘要

Chondroadherin (CHAD) is a cartilage matrix protein that mediates the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine‐rich repeats (LRR) flanked by cysteine‐rich domains. CHAD makes important interactions with collagen as well as with cell‐surface heparin sulfate proteoglycans and α 2 β 1 integrins. The integrin‐binding site is located in a region of hitherto unknown structure at the C‐terminal end of CHAD. Peptides based on the C‐terminal human CHAD (hCHAD) sequence have shown therapeutic potential for treating osteoporosis. This article describes a still‐unconventional structure solution by phasing with de novo models, the first of a β‐rich protein. Structure determination of hCHAD using traditional, though nonsystematic, molecular replacement was unsuccessful in the hands of the authors, possibly owing to a combination of low sequence identity to other LRR proteins, four copies in the asymmetric unit and weak translational pseudosymmetry. However, it was possible to solve the structure by generating a large number of de novo models for the central LRR domain using Rosetta and multiple parallel molecular‐replacement attempts using AMPLE . The hCHAD structure reveals an ordered C‐terminal domain belonging to the LRRCT fold, with the integrin‐binding motif (WLEAK) being part of a regular α‐helix, and suggests ways in which experimental therapeutic peptides can be improved. The crystal structure itself and docking simulations further support that hCHAD dimers form in a similar manner to other matrix LRR proteins.