Molecular Mechanism of Flexible Carbohydrate-Protein Interaction in CD44

描述(申请人提供)：建议研究的目的是通过促进对CD44：透明质酸蛋白：碳水化合物结合作用的理解，为合理开发具有侵袭/转移癌症和炎症性疾病潜在治疗价值的类药物小分子化合物奠定基础。这种结合相互作用是抗癌和抗炎治疗的有效靶点，一直是核磁共振和X射线晶体结构生物学研究的主题，这些研究导致了原子分辨模型。然而，结合位点的构象灵活性、大的蛋白质-碳水化合物结合界面以及变构位点上的有序到无序转变都是使在原子细节水平上理解结合的尝试复杂化的因素。这一结果对于理解CD44的分子基础：透明质酸分子识别和基于结构的CD44类药物抑制物的发现：透明质酸结合都是一个关键的障碍。为了克服这一障碍，将应用原子详细的分子模拟来实现三个具体目标：1.确定结合部位灵活性对结合亲和力的贡献；2.寻找大结合界面上对结合亲和力贡献大于预期的“热点”；3.确定变构部位有序到无序转变对结合部位灵活性和亲和力的贡献。研究设计包括系统地确定弱和动态蛋白质的构象图景的能量学：碳水化合物相互作用，其作用尚未通过实验或计算方法广泛探索。所使用的方法是全原子显式溶剂分子动力学模拟，包括有偏采样和计算炼金术方法，提供关于CD44：透明质酸络合物构象和组成变化的自由能后果的信息。实现上述目标将导致对(A)结合位点的灵活性和亲和力，以及(B)从有序到无序的转变如何影响灵活性和亲和力的原子分辨理解，从而加深对CD44的基本理解：透明质酸分子识别及其在基于结构的CD44：HA抑制剂发现中的应用。) 公共卫生相关性：CD44蛋白和透明质酸碳水化合物的结合作用在某些侵袭性/转移性癌症和炎症性疾病中发挥作用。因此，以这种相互作用为目标可能是帮助治疗这些疾病的一种新方法。为了实现这一新疗法的目标，同时也为了加强对蛋白质中分子灵活性的有限了解：碳水化合物复合体，本工作试图通过应用尖端的计算机模拟方法来加强对这种结合作用的基本分子水平的理解。

DESCRIPTION (provided by applicant): The purpose of the proposed research is to strengthen the foundation for the rational development of drug-like small-molecule compounds with potential therapeutic value in invasive/metastatic cancers and inflammatory disorders by advancing the understanding of the CD44:hyaluronan protein:carbohydrate binding interaction. This binding interaction, which is a validated target for anti-cancer and anti-inflammatory therapy, has been the subject of both NMR and X-ray crystallography structural biology studies that have led to atomic-resolution models. However, conformational flexibility of the binding site, a large protein:carbohydrate binding interface, and an order-to-disorder transition at an allosteric site are all factors that complicate attempts to understand binding at an atomic level of detail. The result is a critical barrier to progress both with respect to understanding the molecular basis for CD44:hyaluronan molecular recognition and the structure-based discovery of drug-like inhibitors of CD44:hyaluronan binding. Toward overcoming this barrier, atomically- detailed molecular simulations will be applied to achieve three specific aims: 1. Determining the contribution of binding site flexibility to binding affinity, 2. Looking for "hot spots" on the large binding interface that make greater than expected contributions to binding affinity, and 3. Determining the contribution of the allosteric site order-to-disorder transition on binding site flexibility and affinity. The research design involves systematically determining the energetics of the conformational landscape underlying weak and dynamic protein:carbohydrate interactions, the role of which has not been explored extensively via either experimental or computational approaches. The methodology to be used is all-atom explicit-solvent molecular dynamics simulations, including biased sampling and computational alchemy approaches that provide information regarding the free-energy consequences of conformational and compositional changes in the CD44:hyaluronan complex. Achieving the stated aims will lead to atomic- resolution understandings of (a) binding site flexibility and affinity, and (b) how flexibility and affinity are impacted by the order-to-disorder transition, thereby furthering the fundamental understanding of CD44:hyaluronan molecular recognition and its application to structure-based discovery of CD44:HA inhibitors.) PUBLIC HEALTH RELEVANCE: The binding interaction of the CD44 protein and the hyaluronan carbohydrate plays roles in certain invasive/metastatic cancers and inflammatory disorders. Targeting this interaction may therefore be a new way to help treat these diseases. Toward this goal of new therapeutics, and also to enhance the limited understanding of molecular flexibility in protein:carbohydrate complexes, the present work seeks to strengthen the fundamental molecular-level understanding of this binding interaction through the application of cutting- edge computer simulation methods.