Hatta T, Konishi H, Katoh E, Natsume T, Ueno N, Kobayashi Y, Yamazaki T
Structural Biology Unit, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Biopolymers. 2000;55(5):399-406. doi: 10.1002/1097-0282(2000)55:5<399::AID-BIP1014>3.0.CO;2-9.
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) >> T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.
骨形态发生蛋白（BMPs）属于多功能细胞因子的转化生长因子-β（TGF-β）超家族。BMP在与细胞表面两种不同的I型和II型受体形成三聚体复合物后，诱导其信号来调节各种细胞的生长、分化和凋亡：这两种受体均为单跨膜丝氨酸/苏氨酸激酶受体。为了鉴定BMP I型受体上负责其配体结合的氨基酸残基，通过丙氨酸扫描诱变研究了BMP IA型受体（sBMPR-IA）细胞外配体结合域的构效关系。突变受体以及sBMPR-IA在大肠杆菌中作为与硫氧还蛋白的融合蛋白表达，并在用肠激酶消化后通过反相高效液相色谱（RP-HPLC）纯化。通过圆二色光谱（CD）对溶液中的亲本蛋白和代表性突变体进行结构分析，结果显示它们的折叠结构没有可检测到的差异。通过表面等离子体共振生物传感器测定突变体与BMP-4的结合亲和力。除Y70A外，所有检测的突变受体对BMP-4的亲和力均降低，亲和力降低的顺序为：I52A（17倍）≈F75A（15倍）>>T64A（4倍）=T62A（4倍）≈E54A（3倍）。后三个突变体观察到的结合亲和力降低主要是由于缔合速率常数降低，而前两个突变体缔合和解离速率常数的改变均导致亲和力大幅降低。这些结果使我们能够得出结论，sBMPR-IA利用分子的凹面识别配体。BMP IA型受体的主要配体结合位点由环2中的苯丙氨酸75以及三链β折叠上的异亮氨酸52、谷氨酸54、苏氨酸62和苏氨酸64组成。这些发现应为TGF-β超家族中配体/I型受体识别提供一般基础。
