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

在这个研究项目中，人类信号酶的三维结构，分子相互作用和灵活性将以最有利于药物发现的分辨率进行表征。这种40 kDa的激酶被称为Ca 2 +/钙调蛋白依赖性蛋白激酶I δ（CaMK 1D），并将磷酸盐添加到丝氨酸或苏氨酸残基上的底物蛋白。我们的蛋白质靶标最近被发现与乳腺细胞的转化和侵袭性密切相关。它的基因在乳腺癌的基底层细胞中经常扩增和过度活跃，导致细胞在不受控制的情况下生长和分裂，这表明CaMK 1D抑制剂可能与改善人类健康和福祉直接相关。然而，这些目标在生理条件下的溶液结构和构象动力学方面的分析仍然非常具有挑战性，需要进一步的基础研究和技术发展，使它们更适合实验研究，并在预测水平上揭示其机制。我们将使用一种称为核磁共振光谱的方法，使用我们国家设施的超导磁体，它可以用来检测大分子中数千个原子核的独特信号。该方法提供了前所未有的信息水平的形状，构象，运动和化学相互作用的蛋白质在三维空间和时间范围从皮秒到秒。我们已经分配了大多数蛋白质的骨架信号，现在计划将这些信号扩展到其解析的复合物和侧链，以比任何其他蛋白激酶更好地了解其分子功能。我们将研究CaMK 1D与其他激活酶的蛋白质以及代谢物和抑制剂的相互作用，我们最近发现这些蛋白质是配体，并开始绘制CaMK 1D的相互作用。我们还预测了一个可以结合脂质和膜的新位点，并将使用自旋标记分子和计算机方法来验证和定义这种拟议机制的性质，以将蛋白质定位到其在细胞中的活性位点。与我们的合作者一起，我们将提供第一个全面的结构，功能和化学见解，了解这种激酶如何在分子水平上发挥作用和发出信号，使我们能够更准确地操纵其在体外和体内的行为。该项目的终点包括与调节其在细胞中活性的脂质，代谢物，底物和蛋白质配体结合的酶的结构，对这些结合事件的动力学和动力学的更深入了解，以及设计抑制剂和突变的合理基础，用于体外和体内分析这种新兴的药物发现靶点。

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.