Structure of Sclerostin Protein Complexes

硬化素蛋白复合物的结构

• 批准号: 8232023
• 负责人: RAJIV KUMAR
• 金额: $ 17.74万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232023
• 关键词: Affect; Affinity; Aging; Alkaline Phosphatase; American; Amino Acids; Anabolic Agents; Apoptosis; Binding; Binding Proteins; Bone Density; C-terminal; Caspase; Cell Differentiation process; Cell Proliferation; Cell division; Complex; Crystallization; Crystallography; Cystine; Data; Development; Disease; Docking; Embryo; Extracellular Domain; Extracellular Matrix Proteins; Fibroblasts; Fracture; Genes; Health; Human; Knowledge; LDL-Receptor Related Protein 1; Link; Mammalian Cell; Mediating; Medical; Mesenchymal Stem Cells; Molecular; Molecular Conformation; Morbidity - disease rate; Mutation; Names; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Postmenopause; Proteins; Reporting; Role; Sclerosis; Signal Transduction; Site; Skeleton; Solutions; Structure; Surface Plasmon Resonance; Syndrome; Teriparatide; Therapeutic; Van Buchem disease; Vascular Endothelial Cell; Woman; Xenopus; angiogenesis; base; bone; bone mass; bone morphogenetic protein 6; bone morphogenetic protein receptors; bone quality; cell motility; cyr61 protein; design; insight; lipoprotein receptor related protein 5; men; mineralization; mutant; protein complex; public health relevance; receptor; receptor binding; small molecule

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.

描述（由申请人提供）：本R21申请的目的是在原子水平上研究硬化蛋白的作用机制，硬化蛋白是一种骨细胞衍生的分泌的胱氨酸结蛋白，通过检查硬化蛋白如何与在调节其活性中起重要作用的蛋白质相互作用来抑制骨形成。因为破坏硬化蛋白的表达或活动会增加骨量，通过我们的研究获得的见解可能有助于设计骨质疏松症的治疗方法，骨质疏松症影响着1000万美国人。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）是据报道介导硬化蛋白活性的关键硬化蛋白相关蛋白。这些蛋白质复合物没有结构数据。此外，初步对接研究表明，在两种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，与其在溶液状态下高度无序的结构相反，当硬化蛋白与BMP6、Cyr61和LRP5等蛋白结合时，硬化蛋白的“环路2”区域（氨基酸G86-R109）变得高度结构化。如果这是真的，硬化蛋白活性可能通过变构调节和靶向其相互作用位点，通过使用口服有效的小分子，在患者中产生新骨来拮抗。在Aim 1中，我们将确定硬化蛋白与BMP6相互作用的分子机制；在Aim 2中，我们将确定硬化蛋白与Cyr61 c端结构域相互作用的分子机制，在Aim 3中，我们将确定硬化蛋白复合物与LRP5细胞外结构域的结晶条件。在Aims 1-2中，我们提出的硬化蛋白相互作用机制将通过x射线晶体学进行探讨，然后通过使用表面等离子体共振分析结构引导突变体的结合亲和力。在Aim 3中，硬化蛋白lrp5的结晶条件为1 ？——建设螺旋桨综合体。意义：骨质疏松症是一种与骨折相关的重要医学健康问题，发病率相当高，尤其在老年和绝经后妇女中普遍存在。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。目前唯一可用的合成代谢药物是特立帕肽，必须通过肠外给药。硬化蛋白是一种抑制骨形成的骨细胞衍生蛋白。拮抗其功能是增加高质量骨的潜在治疗策略。我们的研究将允许在原子水平上理解硬化蛋白和介导硬化蛋白功能的三种蛋白伙伴之间的界面。我们预计这一知识将为开发可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蛋白可能受小分子变构调节的影响。