Structural analysis of BMP and BMP receptor

骨形态发生蛋白（BMPs）调节各种细胞的生长、分化和凋亡。为了阐明BMP的信号转导机制，我们分析了BMP受体的结构和相互作用。小鼠BMP - ia型受体胞外结构域与硫氧还蛋白在大肠杆菌中融合表达。利用异核多维核磁共振方法完成了BMP受体的赋值，确定了BMP受体的溶液结构。换句话说，我们已经获得了在原子分辨率上研究BMP-BMP受体复合物的强大工具和方法。在此基础上，我们设计并合成了几种BMP受体突变体，以鉴定其配体（即BMP）结合位点。突变体受体的CD光谱表明，所有突变体基本上都具有天然的折叠结构。利用几种物理化学方法、表面等离子体共振传感器（BIACORE）、沉降平衡和核磁共振（NMR）测量突变体对BMP-4的亲和力。结合研究表明，三链β-sheet表面的Ile52、Glu54、Thr62和Thr64侧链与环区Phe75形成高亲和力的配体结合面。这些结果为BMP受体识别配体的结构基础提供了更深入的认识。

Bone morphogenetic proteins (BMPs) regulate the growth, differentiation and apoptosis of various cells. To elucidate the mechanisms of signal transduction by BMP, we analyzed the structure and interactions of BMP receptor. The extracellular domain of the mouse BMP type-IA receptor was expressed as a fusion protein with thioredoxin in Escherechia coil. Using heteronuclear multidimensional NMR methods, the assignments of the BMP receptor were completed and the solution structure of BMP receptor was determined. In other words, we have obtained powerful tools and methods to investigate the BMP-BMP receptor complex at atomic resolution. Based upon the resulting structure, we have designed and synthesized several mutants of BMP receptor to identify its ligand (i.e. BMP) binding site. CD spectra of the mutant receptors indicated that all the mutants have essentially the native folding structures. Affinities of the mutants to BMP-4 were measured using several physico-chemical methods, surface plasmon resonance sensor (BIACORE), sedimentation equilibrium and NMR.Binding studies have shown that the side chains of Ile52, Glu54, Thr62 and Thr64 at the surface of the three-stranded β-sheet form a high affinity ligand-binding surface together with Phe75 in loop region. These results provide deeper insights for the structural basis of the ligand recognition by BMP receptor.