Crystal and solution structures of PDE2A and PDE1A

心功能调节的两个最重要的第二信使是环核苷酸cAMP和cGMP。这些分子共同作用来调节钙，从而调节收缩的速度和力量。在心肌细胞中，cAMP和cGMP的一个主要控制点是环核苷酸磷酸二酯酶2A(PDE2A)水平。在心脏基质细胞中，主要的PDE是PDE1a，一种钙/钙调蛋白调节酶。这些酶作为细胞内cGMP的受体和调节器，以及细胞内cAMP和cGMP水平的调节器。在PDE2上发现了两个cGMP的调节变构结合位点，现在被认为是N-末端的GAF结构域。CGMP与这些结构域的结合“激活”了酶的催化域。然而，除了与其他酶类似，人们对这些调节域的三维结构和它们调节催化域的分子机制知之甚少。在PDE1A中，N-末端结构域也调节催化活性，但在这种情况下，调节因子是Ca++/CaM。同样，关于三维结构或CaM的结合如何解除酶的抑制，目前还不清楚。在本申请的第一部分，我们建议通过X射线结晶学和核磁共振技术来确定PDE2A调节的GAF结构域的一级结构。我们还建议使用这些信息以及通过直接结合和突变获得的信息来探索配体占据允许GAF结构域调节酶催化活性的分子机制。在中试实验中，我们首次能够为PDE2的cGMP结合的GAF结构域建立大规模的细菌表达和纯化方案。我们还为来自Sf9细胞的PDE1A和2A全酶制定了较小但足够的表达方案。更重要的是，最近，我们已经能够确定可重复产生GAF域晶体的条件，该晶体的衍射率约为3.0埃。我们在这里建议在存在和不存在调节配体的情况下改进这个GAF结构域的结构，以获得更高的分辨率。由于到目前为止我们只能获得GAF结构域配体cGMP的晶体，我们进一步建议在完成PDE2AGAF结构域的初始结构后，用核磁共振技术确定非配体GAF结构域的三维结构，我们打算用类似的方法将它们与其他含有PDE、PDE5A、PDE10A和PDE11A的GAF结构域的cGMP结合GAF结构域的结构进行比较。在第二部分中，我们建议确定依赖于钙/钙调素的PDE1a的催化域的晶体结构和分离的PDRE1a的CaM结合/抑制域的溶液结构。这种主要在心脏间质细胞中表达的酶被认为调节cAMP和cGMP池，从而影响基质沉积。这种酶是钙离子和环核苷酸通路之间串扰的关键点。我们最近设计了一种PDE1a催化亚基的分离和纯化程序，并获得了衍射率约为8埃的小晶体。我们建议生产更好的晶体并优化这种PDE的结构。最后，我们建议通过一系列的核磁共振结构测定和互补的结合/功能实验来确定PDE1A的抑制结构域与催化亚基相互作用的位置和机制。

Two of the most important regulatory second messengers for cardiac function are the cyclic nucleotides, cAMP and cGMP. These molecules work together to regulate calcium and thereby the rate and force of contraction. In the cardiocyte, a major control point for cAMP and cGMP is at the level of cyclic nucleotide phosphodiesterase 2A (PDE2A). In cardiac stromal cells the major PDE is PDE1A, a calcium/calmodulin regulated enzyme. These enzymes serve as an intracellular receptor and regulators for cGMP and a regulator for both cAMP and cGMP levels in the cell. Two regulatory allosteric binding sites for cGMP are found on PDE2 in what are now recognized as N-terminal GAF domains. Binding of cGMP to these domains "activate" the catalytic domain of the enzyme. However, very little is known about the 3 dimensional structure of these regulatory domains nor the molecular mechanisms by which they regulate the catalytic domain except by analogy to other enzymes. In PDE1A, the N-terminal domain also regulates catalytic activity but in this case the regulator is Ca++/CaM. Again, nothing is known about the 3 dimensional structure or mechanistically how binding of CaM relieves inhibition of the enzyme. In the first part of this application we propose to determine by X-ray crystallographic and nuclear magnetic resonance techniques the primary structure of the regulatory GAF domains of PDE2A. We also propose to use this information along with that obtained from direct binding and mutagenesis to explore the molecular mechanism by which ligand occupancy allows the GAF domains to regulate catalytic activity of the enzyme. In pilot experiments we have been able for the first time to establish large-scale bacterial expression and purification protocols for the cGMP binding GAF domains of PDE2. We have also worked out smaller but adequate expression protocols for the PDE1A and 2A holoenzymes from Sf9 cells. More importantly, very recently, we have been able to determine conditions for reproducibly producing crystals for the GAF domain that diffract to approximately 3.0 angstroms. We are proposing here to refine the structure of this GAF domain to higher resolution in both the presence and absence of regulatory ligand. As we have so far only been able to obtain crystals in the prese3nce of the GAF domain ligand, cGMP, we further propose to determine the three dimensional structure of the non-liganded GAF domain by NMR techniques After completing the initial structures of the PDE2A GAF domains, we intend to use similar methods to compare them to the structure of the cGMP binding GAF domains of the other GAF domain containing PDEs, PDE5A, PDE10A and PDE11A. In the second part, we propose to determine the crystal structure of the catalytic domain of the cardiac Ca2+/CaM dependent PDE1A and the solution structure of the isolated CaM-binding/inhibitory domain of PDRE1A. This enzyme, which is expressed predominantly in stromal cells of the hear5t, is thought to regulate pools of cAMP and cGMP that effect matrix deposition. This enzyme serves as a pivotal point of cross talk between the Ca2+ and cyclic nucleotide pathways. We have recently worked out an isolation and purification procedure for the catalytic subunit of PDE1A and have obtained small crystals that diffract to approximately 8 angstroms. We propose to produce better crystals and refine the structure of this PDE. Finally, we propose to determine the sites and mechanisms by which the inhibitory domain of PDE1A interacts with the catalytic subunit by a series of NMR structural determinations and by complementary binding/function experiments.