Yin Huiran, Yeh Lee-Chuan C, Hinck Andrew P, Lee John C
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
J Mol Biol. 2008 Apr 18;378(1):191-203. doi: 10.1016/j.jmb.2008.02.031. Epub 2008 Mar 4.
ActR-IIA, ActR-IIB, and BMPR-II are low-affinity type II receptors that bind bone morphogenetic proteins (BMPs) in the same overall manner. The binding of BMPs by ActR-IIs has been analyzed structurally and functionally, but no detailed analysis of BMPR-II has been reported. The objective of this study was to determine ligand-binding epitopes and specificity determinants in two regions, the hydrophobic patch and the A-loop of the BMPR-II extracellular domain (ECD). A series of alanine-substituted variants was generated using a recently published X-ray structure of the unliganded form of the ovine BMPR-II ECD as a guide. These variants were characterized using one-dimensional NMR and functional activity assays with BMP-2, BMP-7 and GDF-5 as ligands. The results showed that alanine substitutions of conserved residues W85 and Y113 within the hydrophobic patch of the ECD differentially perturbed BMP ligand binding without disrupting receptor folding, suggesting that they are critical determinants for ligand binding and ligand specificity. Our results further revealed that the nonconserved residue L69 in the hydrophobic patch contributes to ligand-binding activity and specificity. Mutations of several residues within the A-loop resulted in minimal effects on the binding of the different BMP ligands. Overall, these observations identify several amino acid residues that play different roles in BMPR-II and ActR-II and thereby enable BMPR-II and ActR-IIs to bind different subclasses of BMP ligands.
激活素受体IIA（ActR-IIA）、激活素受体IIB（ActR-IIB）和骨形态发生蛋白受体II（BMPR-II）是低亲和力的II型受体，它们以大致相同的方式结合骨形态发生蛋白（BMP）。激活素受体II（ActR-IIs）与BMP的结合已在结构和功能上进行了分析，但尚未见有关BMPR-II的详细分析报道。本研究的目的是确定BMPR-II细胞外结构域（ECD）的两个区域，即疏水斑块和A环中的配体结合表位和特异性决定因素。以最近发表的绵羊BMPR-II ECD未结合配体形式的X射线结构为指导，生成了一系列丙氨酸取代变体。使用一维核磁共振以及以BMP-2、BMP-7和生长分化因子5（GDF-5）作为配体的功能活性测定对这些变体进行了表征。结果表明，ECD疏水斑块内保守残基W85和Y113的丙氨酸取代对BMP配体结合有不同程度的干扰，但不破坏受体折叠，这表明它们是配体结合和配体特异性的关键决定因素。我们的结果进一步表明，疏水斑块中的非保守残基L69有助于配体结合活性和特异性。A环内几个残基的突变对不同BMP配体的结合影响最小。总体而言，这些观察结果确定了几个在BMPR-II和ActR-II中发挥不同作用的氨基酸残基，从而使BMPR-II和ActR-II能够结合不同亚类的BMP配体。
