Groppe Jay, Greenwald Jason, Wiater Ezra, Rodriguez-Leon Joaquin, Economides Aris N, Kwiatkowski Witek, Baban Kandan, Affolter Markus, Vale Wylie W, Izpisua Belmonte Juan Carlos, Choe Senyon
Salk Institute, La Jolla, CA 92037, USA.
J Bone Joint Surg Am. 2003;85-A Suppl 3:52-8. doi: 10.2106/00004623-200300003-00010.
The activity of bone morphogenetic proteins (BMPs) is regulated extracellularly by several families of secreted, negatively-acting factors. These BMP antagonists participate in the control of a diverse range of embryonic processes, such as establishment of the dorsal-ventral axis, neural induction, and formation of joints in the developing skeletal system. The ongoing process of neurogenesis in the adult brain also requires inhibition of BMP ligand activity. To date, the three-dimensional structures of these antagonists as well as the nature of their interaction with ligand have remained unknown. Toward that end, we have determined the crystal structure of the antagonist Noggin bound to BMP-7.
The complex of the two homodimeric proteins was preformed, isolated by size exclusion chromatography, and crystallized at neutral pH. To probe the molecular interface of the complex and to quantitate the activity of a human mutant form, variant Noggin proteins were produced and their binding affinities were measured in vitro. The correlation between binding affinity and biological activity was examined with Noggin-soaked beads implanted in the developing chick limb bud.
The structure of the complex reveals that Noggin inhibits BMP signaling by blocking the binding sites of both types of receptors (Type I and Type II), mimicking their modes of binding. The affinity of Noggin variants for BMP-7 correlated well with the inhibition of BMP-induced chondrogenesis in the chick limb bud, confirming that Noggin acts by sequestering the ligand in an inactive state. Interestingly, the scaffold of Noggin was found to contain a cystine knot topology and protein fold similar to that of BMPs, indicating that ligand and antagonist may have evolved from a common ancestral gene.
骨形态发生蛋白（BMPs）的活性在细胞外受到几类分泌型负向作用因子家族的调控。这些BMP拮抗剂参与多种胚胎发育过程的控制，如背腹轴的建立、神经诱导以及发育中的骨骼系统关节的形成。成人大脑中正在进行的神经发生过程也需要抑制BMP配体的活性。迄今为止，这些拮抗剂的三维结构以及它们与配体相互作用的性质仍然未知。为此，我们确定了与BMP-7结合的拮抗剂Noggin的晶体结构。
两种同二聚体蛋白的复合物预先形成，通过尺寸排阻色谱法分离，并在中性pH下结晶。为了探究复合物的分子界面并定量一种人类突变体形式的活性，制备了变体Noggin蛋白，并在体外测量它们的结合亲和力。通过将浸泡有Noggin的珠子植入发育中的鸡胚肢芽来检查结合亲和力与生物活性之间的相关性。
复合物的结构表明，Noggin通过阻断两种类型受体（I型和II型）的结合位点来抑制BMP信号传导，模仿它们的结合模式。Noggin变体对BMP-7的亲和力与鸡胚肢芽中BMP诱导的软骨生成抑制密切相关，证实Noggin通过将配体隔离在无活性状态下起作用。有趣的是，发现Noggin的支架含有与BMPs相似的胱氨酸结拓扑结构和蛋白质折叠，表明配体和拮抗剂可能从一个共同的祖先基因进化而来。
