Molecular mechanisms of calcium/calmodulin-dependent kinase localisation activation and inhibition

钙/钙调蛋白依赖性激酶定位激活和抑制的分子机制

• 批准号: BB/H019383/1
• 负责人: Michael Overduin
• 金额: $ 50.73万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH019383%2F1
• 关键词: Molecular; mechanisms; calcium; calmodulin; dependent

In this research project the three dimensional structures, and molecular interactions and flexibility of a human signaling enzyme will be characterized at a resolution that is most useful for drug discovery. This 40 kDa kinase is known as Ca2+/calmodulin-dependent protein kinase I delta (CaMK1D), and adds phosphates to substrate proteins on serine or threonine residues. Our protein target has recently been found to be critically involved in the transformation and invasiveness of breast cells. Its gene is frequently amplified and hyperactive in basal layer cells of breast carcinomas, causing cells to grow and divide without control, and suggesting that CaMK1D inhibitors could have direct relevance to improving human health and well being. However, these targets still remain very challenging for analysis in terms of their solution structures and conformational dynamics under physiological conditions, warranting further fundamental research and technological developments to render them more amenable to experimental investigation and uncover their mechanisms at a predictive level. We will use a method known as nuclear magnetic resonance spectroscopy using our national facility's superconducting magnets, which can be used to detect a unique signal for the individual thousands of atomic nuclei in the macromolecule. The method provides an unprecedented level of information about the shape, conformation, motions and chemical interactivity of a protein in three dimensional space and over a range of timescales from picoseconds to seconds. We have assigned most of the protein's backbone signals, and now plan to extend these to its resolved complexes and sidechains to understand its molecular functions better than any other protein kinase. We will study the interactions of CaMK1D with other proteins that activate the enzyme as well as metabolites and inhibitors which we have recently discovered as ligands and begun to map the interactions of. We have also predicted a novel site that could bind lipids and membranes, and will use spin label molecules and computer methods to validate and define the nature of this proposed mechanism to localize the protein to its sites of activity in cells. Together with our collaborators we will provide the first comprehensive structural, functional and chemical insights into how this kinase acts and signals at a molecular level, allowing us to much more accurately manipulate its behaviour in vitro and in vivo. The endpoints of the project include structures of the enzyme bound to the lipids, metabolites, substrates and protein ligands that regulate its activity in cells, a deeper understanding of the dynamics and kinetics of these binding events, and a rational basis for designing inhibitors and mutations for in vitro and in vivo analysis of this emerging target for drug discovery.

在这项研究项目中，将以最有用的分辨率来表征三维结构，人类信号传导酶的分子相互作用和灵活性。该40 kDa激酶被称为Ca2+/钙调蛋白依赖性蛋白激酶I Delta（CAMK1D），并将磷酸盐添加到丝氨酸或苏氨酸残基上的底物蛋白中。最近，我们发现我们的蛋白质靶标与乳腺细胞的转化和侵入性非常重要。它的基因在乳腺癌的基底层细胞中经常被扩增和过度活跃，导致细胞在没有控制的情况下生长和分裂，并表明CAMK1D抑制剂可能与改善人类健康和健康有直接相关。但是，这些靶标在生理条件下的溶液结构和构象动态方面仍然非常具有挑战性，需要进一步的基本研究和技术发展，以使它们更适合实验研究并在预测水平上揭示其机制。我们将使用我们国家设施的超导磁体使用一种称为核磁共振光谱的方法，该磁体可用于检测大分子中数千个原子核的独特信号。该方法提供了关于蛋白质在三维空间中以及从picseconds到秒的一系列时间表的形状，构象，运动和化学相互作用的前所未有的信息。我们已经分配了大多数蛋白质的主链信号，现在计划将其扩展到其分辨的复合物和侧链，以比任何其他蛋白激酶更好地理解其分子功能。我们将研究CAMK1D与激活酶以及代谢物和抑制剂的其他蛋白质的相互作用，这些蛋白质以及我们最近以配体发现并开始绘制相互作用的代谢产物和抑制剂。我们还预测了一个可以结合脂质和膜的新型位点，并将使用自旋标记分子和计算机方法来验证和定义该提出的机制的性质，以将蛋白质定位于细胞中的活性位点。与我们的合作者一起，我们将提供第一个综合的结构，功能和化学见解，以了解该激酶在分子水平上的作用和信号，从而使我们能够在体外和体内更准确地操纵其行为。该项目的终点包括与脂质，代谢产物，底物和蛋白质配体结合的结构，这些酶调节其在细胞中的活性，对这些结合事件的动力学和动力学有更深入的了解，以及用于设计抑制剂的合理基础，并针对该抑制剂设计抑制剂，以实现该抑制剂的抑制剂和突变，以实现该剂量和INVIVO分析的药物分析目标。

项目成果

Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection.

• DOI: 10.1074/jbc.m116.722066
• 发表时间: 2016-04-22
• 期刊: The Journal of biological chemistry
• 作者: Salim M;Knowles TJ;Hart R;Mohammed F;Woodward MJ;Willcox CR;Overduin M;Hayday AC;Willcox BE
• 通讯作者: Willcox BE

NMR of Membrane Proteins: Beyond Crystals.

膜蛋白的核磁共振：超越晶体。

• DOI: 10.1007/978-3-319-35072-1_3
• 发表时间: 2016
• 期刊: Advances in experimental medicine and biology
• 作者: Rajesh S

Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

• DOI: 10.3390/cells5020026
• 发表时间: 2016-06-13
• 期刊: Cells
• 影响因子: 6
• 作者: Smithers CC;Overduin M
• 通讯作者: Overduin M

Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting.

• DOI: 10.1038/s41467-018-03370-1
• 发表时间: 2018-03-08
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Lenoir M;Ustunel C;Rajesh S;Kaur J;Moreau D;Gruenberg J;Overduin M
• 通讯作者: Overduin M