Bone morphogenic protein-2 (BMP-2) is the most documented member of BMP family and plays a crucial role in bone formation and growth. In this study, we systematically analyze and compare the complex crystal structures and interaction properties of BMP-2 with its cognate receptors BMPR-I/BMPR-II and with its natural antagonist crossveinless-2 (CV-2) using an integrated in silico-in vitro strategy. It is found that the antagonist-binding site is not fully overlapped with the two receptor-binding sites on BMP-2 surface; the antagonist can competitively disrupt BMP-2–BMPR-II interaction using a blocking-out-of-site manner, but has no substantial influence on BMP-2–BMPR-I interaction. Here, the antagonist-binding site is assigned as a new functional epitope armpit to differ from the traditional conformational epitope wrist and linear epitope knuckle at receptor-binding sites. Structural analysis reveals that the armpit comprises three sequentially discontinuous, structurally vicinal peptide segments, separately corresponding to a loop region and two β-strands crawling on the protein surface. The three segments cannot work independently when splitting from the protein context, but can restore binding capability to CV-2 if they are connected to a single peptide. A systematic combination of different-length polyglycine linkers between these segments obtains a series of designed single peptides, from which several peptides that can potently interact with the armpit-recognition site of CV-2 with high affinity and specificity are identified using energetic analysis and fluorescence assay; they are expected to target BMP-2–CV-2 interaction in a self-inhibitory manner.

骨形态发生蛋白2（BMP-2）是BMP家族中最有据可查的成员，并且在骨形成和生长中起关键作用。在这项研究中，我们使用集成的计算机模拟系统分析和比较了BMP-2及其同源受体BMPR-I / BMPR-II及其天然拮抗剂crossveinless-2（CV-2）的复杂晶体结构和相互作用特性。体外策略。发现拮抗剂结合位点没有与BMP-2表面上的两个受体结合位点完全重叠；相反，结合位点与BMP-2表面上的两个受体结合位点没有完全重叠。该拮抗剂可以通过异位阻断竞争性地破坏BMP-2–BMPR-II的相互作用，但对BMP-2–BMPR-I的相互作用没有实质性影响。在这里，拮抗剂结合位点被指定为新的功能性抗原决定簇腋窝不同于传统的构象表位手腕和受体结合位点的线性表位关节。结构分析表明，腋窝包括三个依次不连续的，结构上相邻的肽段，分别对应于一个环区和两个在蛋白质表面上爬行的β链。从蛋白质环境中分离出来时，这三个片段不能独立发挥作用，但是如果它们连接至单个肽，则可以恢复与CV-2的结合能力。这些区段之间不同长度的聚甘氨酸接头的系统组合，获得了一系列设计的单肽，从中可以与腋下有效相互作用的几种肽用能量分析和荧光分析法鉴定具有高亲和力和特异性的CV-2的识别位点；它们有望以自我抑制的方式靶向BMP-2–CV-2相互作用。