Crystal structure of human chondroadherin: solving a difficult molecular-replacement problem usingde novomodels

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 αβ 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.

Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin

“…While there are contacts between the concave faces in the fibromodulin and chondroadherin crystals, the paired molecules are arranged differently, with the two solenoids crossing at an angle rather than forming the elongated dimers of decorin and biglycan. Notably, the packing of chondroadherin molecules in our crystal structure is different from that seen in another chondroadherin structure [34] , suggesting that the concave face interactions are not physiologically relevant ( Fig. 3 ).…”

“…Comparison of our chondroadherin structure (crystallised at pH 4.8) with the structure recently published (crystallised at pH ~ 10) [34] reveals no noteworthy differences in tertiary structure: the average r.m.s. deviation of the eight pairwise superpositions of crystallographically independent chains is 0.94 ± 0.31 Å (322 Cα atoms).…”

“…3 ). A recent study proposed that chondroadherin may form physiological dimers, but this proposal was based solely on an analysis of crystal lattice interactions and computational docking [34] . Our SEC-MALS experiment ( Fig.…”

“…Even so, further structural studies of the SLRP family are clearly warranted. At the time of writing, the only crystal structures available were those of two closely related class I SLPRs, decorin [30] and biglycan [33] ; a chondroadherin structure was published while this manuscript was in preparation [34] . Here, we report the crystal structure of fibromodulin and a different crystal form of chondroadherin, which reveal that these SLRPs do not self-associate in the same manner as decorin or biglycan.…”

Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats in Bacterial Type III Secretion System Effectors

“…The variable N-type VSs are seen in YopM—RS LC DLPPS and SG LS E LP PN (Supplementary Table S2). The first repeat of twelve LRRs in chondroadherin is also a variable N-subtype VS of QK IP K VS EK; the structure of human chondroadherin which forms tetramers in crystal has been determined at 2.1 Å resolution [ 69 ]. Their secondary structure assignment sometimes shows no PPII or only short PPII of two or three residues.…”