Structure of sclerostin protein complexes

描述(申请人提供)：本R21申请的目标是在原子水平上研究硬化素的作用机制，硬化素是一种源自骨细胞的、分泌的、半胱氨酸结蛋白，通过检测硬化素如何与在调节其活性中起关键作用的蛋白质相互作用来抑制骨形成。由于硬化素表达或活性的中断会增加骨量，通过我们的研究得出的见解可能有助于设计治疗骨质疏松症的方法，骨质疏松症是一种影响1000万美国人的疾病。骨形态发生蛋白6(Bmp6)、富含半胱氨酸的蛋白61(Cyr61)和低密度脂蛋白受体相关蛋白5(LRP5)是已报道的介导硬化素活性的关键硬化素相关蛋白。这些蛋白质复合体没有结构数据。此外，初步的对接研究表明，在两个硬化素核磁共振溶液结构中观察到的构象必须改变才能影响与这三种蛋白质的结合。我们假设，与其在溶液状态下的高度无序结构相反，当skerostin与其调节功能的蛋白质如Bmp6、Cyr61和LRP5结合时，skerostin(氨基酸g86-R109)的“环2”区域变得高度结构化。如果是真的，硬化素的活性可能会被变构调节和靶向其相互作用部位(S)，通过使用口服有效的小分子在患者中产生新骨来拮抗。在目标1中，我们将确定硬化素与Bmp6相互作用的分子机制；在目标2中，我们将确定硬化素与Cyr61 C末端结构域相互作用的分子机制；在目标3中，我们将确定硬化素与LRP5胞外区形成的复合体的结晶条件。在目标1-2中，我们将通过X射线结晶学来探索我们提出的硬化素相互作用的机制，然后通过使用表面等离子体共振来分析结构导向突变体的结合亲和力。在AIM中，将建立3个结晶硬化素-LRP5第一推进器络合物的条件。意义：骨质疏松症是一个与骨折和相当高的发病率相关的重大医疗健康问题，特别是在老年和绝经后妇女中流行。虽然有效的抗骨吸收药物可用于治疗骨质疏松症，但它们对骨形成的影响很小。现在唯一可用的合成代谢药物是必须非肠道给药的Teriparatide。硬化素是一种骨细胞衍生的蛋白质，可以抑制骨形成。拮抗其功能是增加高质量骨的潜在治疗策略。我们的研究将使我们能够在原子水平上了解硬化素与三个介导硬化素功能的蛋白质伙伴之间的界面。我们预计这些知识将为开发可以口服的拮抗药物提供强有力的基础，特别是如果结构结果表明硬化素可能服从小分子的变构调节。 公共卫生相关性：治疗骨质疏松症需要口服有效的造骨药物。通过确定硬化素(一种骨抑制蛋白)与其相互作用的其他蛋白质的复合体的结构，我们将能够获得允许设计此类药物的信息。这一努力将极大地帮助患有骨质疏松症和骨质疏松相关骨折的女性和男性。

DESCRIPTION (provided by applicant): The objective of this R21 application is to investigate at the atomic level, the mechanism of action of sclerostin, an osteocyte-derived, secreted, cystine-knot protein that inhibits bone formation by examining how sclerostin interacts with proteins that play an essential role in mediating its activity. Because the disruption of sclerostin expression or activity increases bone mass insights developed through our studies may be useful in devising treatments for osteoporosis, a disease affecting 10 million Americans. Bone morphogenetic protein 6 (BMP6), cysteine-rich protein 61 (Cyr61) and low-density-lipoprotein receptor-related protein 5 (LRP5) are key sclerostin-associating proteins reported to mediate sclerostin activity. No structural data exists for these protein complexes. Moreover, preliminary docking studies suggest that the conformations observed in two sclerostin NMR solution-structures must be altered to affect binding to these three proteins. We hypothesize that in contrast to its highly disordered structure in the solution-state, the "loop 2" region of sclerostin (amino acids G86-R109) becomes highly structured when sclerostin binds proteins such as BMP6, Cyr61 and LRP5 whose functions it modulates. If true, sclerostin activity might be antagonized by allosteric modulation and by targeting its interaction site(s), through the use of orally effective, small molecules that would generate new bone in patients. In Aim 1 we will determine the molecular mechanism of sclerostin interaction with BMP6; in Aim 2 we will determine the molecular mechanism of sclerostin interaction with Cyr61 C-terminal domain and in Aim 3 we will identify crystallization conditions for the sclerostin complex with LRP5 extracellular domain. In Aims 1-2, our proposed mechanism for sclerostin interaction will be probed by x-ray crystallography followed by analysis of binding-affinities of structure-guided mutants through the use of surface plasmon resonance. In Aim 3 conditions for crystallizing sclerostin-LRP5 1st ?-propeller complexes will be established. Significance: Osteoporosis is a significant medical health problem associated with fractures and considerable morbidity, prevalent particularly in aging and post-menopausal women. While effective bone anti-resorptive drugs are available for osteoporosis treatment, they have little effect on bone formation. The only anabolic agent available now is teriparatide that must be administered parenterally. Sclerostin is an osteocyte derived protein that inhibits bone formation. Antagonizing its function is a potential therapeutic strategy to increase high quality bone. Our studies will allow an atomic-level understanding of the interface between sclerostin and three protein partners that mediate sclerostin function. We anticipate this knowledge would provide a strong basis for development of antagonistic drugs that can be administrated orally, especially if structural results suggest sclerostin might be amenable to allosteric modulation by small-molecules. PUBLIC HEALTH RELEVANCE: Orally effective drugs that build bone are required for the treatment of osteoporosis. By determining the structures of complexes of sclerostin (a bone inhibitory protein) with other proteins with which it interacts, we will be able to obtain the information that will allow the design of such drugs. This effort will greatly help women and men with osteoporosis and osteoporosis-related fractures.