Structure of sclerostin protein complexes

硬化蛋白复合物的结构

• 批准号: 8086488
• 负责人: RAJIV KUMAR
• 金额: $ 21.29万
• 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/8086488
• 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; 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）：该R21申请的目的是在原子水平上研究sclerostin的作用机制，sclerostin是一种骨细胞衍生的分泌型胱氨酸结蛋白，通过检查sclerostin如何与在介导其活性中发挥重要作用的蛋白质相互作用来抑制骨形成。由于sclerostin表达或活性的破坏增加了骨量，因此通过我们的研究开发的见解可能有助于设计骨质疏松症的治疗方法，这种疾病影响了1000万美国人。骨形态发生蛋白6（BMP 6）、富含半胱氨酸的蛋白61（Cyr 61）和低密度脂蛋白受体相关蛋白5（LRP 5）是介导硬化蛋白活性的关键硬化蛋白相关蛋白。没有这些蛋白质复合物的结构数据。此外，初步对接研究表明，在两个sclerostin NMR溶液中观察到的构象结构必须改变，以影响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sclerostin结合其调节功能的蛋白质如BMP 6、Cyr 61和LRP 5时，sclerostin的“环2”区域（氨基酸G86-R109）变得高度结构化。如果是真的，硬化蛋白活性可能通过变构调节和靶向其相互作用位点来拮抗，通过使用口服有效的小分子，这些小分子将在患者中产生新骨。在目标1中，我们将确定sclerostin与BMP 6相互作用的分子机制;在目标2中，我们将确定sclerostin与Cyr 61 C-末端结构域相互作用的分子机制，并且在目标3中，我们将鉴定sclerostin与LRP 5细胞外结构域复合物的结晶条件。在目标1-2中，我们提出的sclerostin相互作用的机制将通过X射线晶体学进行探索，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。在目标3中，用于结晶硬化素-LRP 5 1st？将建立螺旋桨复合体。 重要性：骨质疏松症是一个重要的医学健康问题，与骨折和相当大的发病率，特别是在老年和绝经后妇女中流行。虽然有效的骨抗吸收药物可用于骨质疏松症治疗，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teriparbestrol，必须通过胃肠外途径给药。硬化蛋白是一种骨细胞衍生的蛋白质，抑制骨形成。拮抗其功能是增加高质量骨的潜在治疗策略。我们的研究将允许一个原子水平的理解sclerostin和三个蛋白质的合作伙伴之间的接口，介导sclerostin功能。我们预计这方面的知识将提供一个强有力的基础，可以口服给药的拮抗药物的发展，特别是如果结构的结果表明sclerostin可能会受到小分子变构调制。 公共卫生相关性：治疗骨质疏松症需要口服有效的骨生成药物。通过确定sclerostin（一种骨抑制蛋白）与其他蛋白质相互作用的复合物的结构，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妇女和男子骨质疏松症和骨质疏松症相关的骨折。