Rämisch Sebastian, Pramhed Anna, Tillgren Viveka, Aspberg Anders, Logan Derek T
Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00 Lund, Sweden.
Department of Rheumatology and Molecular Skeletal Biology, Clinical Sciences Lund, Lund University, BMC-C12, SE-221 84 Lund, Sweden.
Acta Crystallogr D Struct Biol. 2017 Jan 1;73(Pt 1):53-63. doi: 10.1107/S205979831601980X.
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.
软骨粘连蛋白（CHAD）是一种介导分离软骨细胞粘附的软骨基质蛋白。其蛋白质核心由11个富含亮氨酸的重复序列（LRR）组成，两侧为富含半胱氨酸的结构域。CHAD与胶原蛋白以及细胞表面硫酸乙酰肝素蛋白聚糖和αβ整合素发生重要相互作用。整合素结合位点位于CHAD C末端迄今未知结构的区域。基于C末端人CHAD（hCHAD）序列的肽已显示出治疗骨质疏松症的潜力。本文描述了一种通过从头模型进行相位分析的非常规结构解析方法，这是首次针对富含β折叠的蛋白质。作者尝试使用传统但非系统的分子置换方法来确定hCHAD的结构，但未成功，这可能是由于与其他LRR蛋白的序列同一性较低、不对称单元中有四个拷贝以及较弱的平移假对称性等多种因素共同作用的结果。然而，通过使用Rosetta为中央LRR结构域生成大量从头模型，并使用AMPLE进行多次平行分子置换尝试，得以解析出其结构。hCHAD结构揭示了一个属于LRRCT折叠的有序C末端结构域，整合素结合基序（WLEAK）是规则α螺旋的一部分，并提出了改进实验性治疗肽的方法。晶体结构本身和对接模拟进一步支持hCHAD二聚体以与其他基质LRR蛋白相似的方式形成。
