COMPUTATIONAL MODELING OF INTERACTIONS BETWEEN HYALURONAN AND LINK MODULES

透明质酸和连接模块之间相互作用的计算模型

• 批准号: 7601409
• 负责人: PAUL L DEANGELIS
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601409
• 关键词: Almond Nut; Arthritis; Binding; Binding Sites; CD44 Antigens; Carbohydrates; Cartilage; Cereals; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Connective and Soft Tissue; Data; Disease; Extracellular Matrix; Facility Construction Funding Category; Funding; Goals; Grant; Homology Modeling; Hyaluronan; Infertility; Institution; Investigation; Ions; Ligands; Link; Macromolecular Complexes; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Conformation; Nuclear Magnetic Resonance; Polymers; Polysaccharides; Process; Property; Proteins; Proteoglycan; Recombinants; Research; Research Personnel; Resolution; Resources; Solutions; Solvents; Source; Structure; TSG-6 protein; Tertiary Protein Structure; Tissues; United States National Institutes of Health; Vertebrates; X-Ray Crystallography; aggrecan; day; insight; interfacial; link protein; molecular dynamics; molecular mass; molecular mechanics; molecular modeling; novel; programs; protein expression; research study; restraint; simulation; versican

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In vertebrates, hyaluronan (HA) is an extracellular matrix polysaccharide that occurs in a free state in soft connective tissue where it conveys its viscoelastic properties and acts as an intercellular spacefiller. HA also interacts with proteins (via Link module domains) and can form large macromolecular complexes that are important in extracellular matrix assembly and remodelling and provide turgor in cartilage and other tissue. At present, the local solution structure of HA is subject to debate and little is known about the three dimensional organisation of the macromolecular HA-protein complexes thus limiting our understanding of the extracellular matrix and various diseases, e.g., arthritis, cancer, and infertility. Recently, we have shown how atomic-scale simulations of HA fragments (e.g., 8 saccharides long) may be used to help interpret nuclear magnetic resonance (NMR) experiments. Simulations have provided much insight into the local conformational state of hyaluronan in solution which is an important determinant for the viscoelastic and hydrodynamic properties of HA. Similarly, a high-resolution structure for a recombinant HA-binding domain (Link module) from the protein TSG-6 (denoted Link_TSG6) in both its HA-bound and un-liganded forms, has been obtained from NMR data. By exclusive use of NMR it has not been possible to characterize the orientation/conformation of the HA ligand de novo. However, the use of experimental data and structural constrains from molecular modeling have facilitated construction of a preliminary model of the binary complex (1). Expression constructs have been produced for other Link module containing proteins, namely the G1-domains of aggrecan and versican, cartilage link protein and link protein 3 that all contain a contiguous pair of Link modules in their HA-binding domains. These protein domains facilitate the attachment of proteoglycans to HA chains, responsible for the formation of large macromolecular aggregates. The interaction is often mediated through a ternary complex between HA, the proteoglycan and a stabilising link protein leading to a structure that is essentially undissociable. Homology modeling, combined with constraints deduced from the likely HA binding site, has allowed models of the double link modules to be constructed for these proteins (2). RESEARCH FOCUS: Our main goals are (a) characterizing the molecular interactions that occur between HA and the variety of Link module containing proteins and (b) determining the three dimensional organisation of supramolecular HA-protein complexes by using novel compounds such as specifically isotopically enriched hyaluronan and specific HA-binding protein expression constructs together with high-field NMR and X-ray crystallography. However, the HA polymer is dynamic and a full understanding of the molecular interactions between HA and the proteins it binds will necessitate an investigation of molecular dynamics. In this regard, we have already modeled the Link module from TSG-6 using an all-atom approach in the presence of explicit solvent and ions and have shown that there is a significant overlap with experimental NMR data. We aim to use computational modeling to aid the process of understanding dynamic interactions between Link modules and HA and their supramolecular organisation. In particular, we intend to model HA-protein complexes using all-atom molecular modelling and eventually coarse-grained molecular modelling. The large molecular complexes that we are aiming to model necessitate more computational resources than we currently have at our disposal. This PSC project has three Specific Aims: I) Molecular modeling of protein-carbohydrate complexes using restraints from newly obtained NMR data. II) All-atom molecular dynamics simulations of protein-carbohydrate complexes using explicit solvent and ions to provide information about interfacial dynamics and to help interpret experimental data. III) Model interactions between multiple proteins and large molecular mass HA using coarse-grained modeling. Computational Requirements: Molecular mechanics minimization/molecular dynamics of 20-30,000 atoms with CHARMM program on Lemieux, Rachel or Jonas. REFERENCES 1. Blundell, C. D.; Mahoney, D. J.; Almond, A.; DeAngelis, P. L.; Kahmann, J. D.; Teriete, P.; Pickford, A. R.; Campbell, I. D.; Day, A. J., J Biol Chem 2003, 278, 49261-70. 2. Blundell, C. D.; Almond, A.; Mahoney, D. J.; DeAngelis, P. L.; Campbell, I. D.; Day, A. J., J Biol Chem 2005, 280, 18189-201.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在脊椎动物中，透明质酸（HA）是一种细胞外基质多糖，以游离状态存在于软结缔组织中，在软结缔组织中，透明质酸传递其粘弹性，并充当细胞间空间填充物。HA还与蛋白质相互作用（通过连接模块结构域），并且可以形成在细胞外基质组装和重塑中重要的大分子复合物，并在软骨和其他组织中提供膨压。目前，HA的局部溶液结构受到争论，并且对大分子HA-蛋白质复合物的三维组织知之甚少，因此限制了我们对细胞外基质和各种疾病的理解，例如，关节炎癌症和不育症最近，我们已经展示了HA片段的原子级模拟（例如，8英寸长）可用于帮助解释核磁共振（NMR）实验。模拟已经提供了很多洞察透明质酸在溶液中的局部构象状态，这是一个重要的决定因素的粘弹性和流体力学性能的HA。类似地，从NMR数据获得了来自蛋白质TSG-6（表示为Link_TSG 6）的HA结合和未配体形式的重组HA结合结构域（Link模块）的高分辨率结构。通过专门使用NMR，不可能从头表征HA配体的取向/构象。然而，使用实验数据和分子模拟的结构约束，促进了二元复合物（1）的初步模型的构建。已经产生了其它含有连接模块的蛋白质的表达构建体，即聚集蛋白聚糖和多功能蛋白聚糖的G1结构域、软骨连接蛋白和连接蛋白3，它们都在其HA结合结构域中含有一对连续的连接模块。这些蛋白质结构域促进蛋白聚糖与HA链的连接，负责形成大的大分子聚集体。这种相互作用通常通过HA、蛋白聚糖和稳定剂之间的三元复合物介导。 连接蛋白 导致基本上不可分离的结构。同源性建模，结合从可能的HA结合位点推导出的约束条件，已经允许为这些蛋白质构建双连接模块的模型（2）。研究重点：我们的主要目标是（a）表征HA和包含蛋白质的各种连接模块之间发生的分子相互作用，和（B）通过使用新化合物（例如特异性同位素富集的透明质酸和特异性HA结合蛋白表达构建体）连同高场NMR和X射线晶体学来确定超分子HA-蛋白质复合物的三维组织。然而，HA聚合物是动态的，要充分了解HA与其结合的蛋白质之间的分子相互作用，就必须研究分子动力学。在这方面，我们已经使用全原子方法在明确的溶剂和离子的存在下对TSG-6的Link模块进行了建模，并表明与实验NMR数据有显著的重叠。我们的目标是使用计算建模，以帮助了解链接模块和HA及其超分子组织之间的动态相互作用的过程。特别是，我们打算模型HA-蛋白质复合物使用全原子分子建模，并最终粗粒度的分子建模。我们的目标是模拟大分子复合物，这需要比我们目前拥有的更多的计算资源。这个PSC项目有三个具体目标：I）使用新获得的NMR数据的限制进行蛋白质-碳水化合物复合物的分子建模。II）使用显式溶剂和离子对蛋白质-碳水化合物复合物进行全原子分子动力学模拟，以提供有关界面动力学的信息并帮助解释实验数据。III）使用粗粒度建模对多种蛋白质和大分子量HA之间的相互作用进行建模。计算要求：分子力学最小化/分子动力学的20- 30000原子与CHARMM程序Lemieux，雷切尔或乔纳斯。参考资料布伦德尔角D.的; Mahoney，D. J.道：Almond，A.; DeAngelis，P. L.; Kahmann，J. D.; Teriete，P.; Pickford，A.的R.;坎贝尔岛D.的; Day、A. J.，J Biol Chem 2003，278，49261-70. 2.布伦德尔，C。D.的; Almond，A.; Mahoney，D. J.道：DeAngelis，P. L.;坎贝尔岛D.的; Day、A. J.，J Biol Chem 2005，280，18189-201。