title: Crystal structure of human chondroadherin: solving a difficult molecular-replacement problem using de novo models.
abstract: 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 α2β1integrins. 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.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00 Lund, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rämisch
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00 Lund, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Pramhed
authors.affiliation: Department of Rheumatology and Molecular Skeletal Biology, Clinical Sciences Lund, Lund University, BMC-C12, SE-221 84 Lund, Sweden.
authors.forename: Viveka
authors.initials: V
authors.lastname: Tillgren
authors.affiliation: Department of Rheumatology and Molecular Skeletal Biology, Clinical Sciences Lund, Lund University, BMC-C12, SE-221 84 Lund, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Aspberg
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00 Lund, Sweden.
authors.forename: Derek T
authors.initials: DT
authors.lastname: Logan
keywords: chondroadherin; collagen binding; de novo models; extracellular matrix; integrin binding; molecular replacement; small leucine-rich repeat proteins; structure prediction