Crystal and solution structures of PDE2A and PDE1A

两个最重要的调节心脏功能的第二信使是环核苷酸，cAMP和cGMP。 这些分子共同调节钙，从而调节收缩的速度和力量。在心肌细胞中，cAMP和cGMP的主要控制点在环核苷酸磷酸二酯酶2A（PDE 2A）的水平。在心脏基质细胞中，主要PDE是PDE 1A，一种钙/钙调蛋白调节酶。这些酶作为cGMP的细胞内受体和调节剂以及细胞中cAMP和cGMP水平的调节剂。在PDE 2上发现了cGMP的两个调节变构结合位点，其现在被认为是N-末端GAF结构域。cGMP与这些结构域的结合“激活”酶的催化结构域。然而，很少有人知道这些调控结构域的三维结构，也不是它们调节催化结构域的分子机制，除了通过类推到其他酶。 在PDE 1A中，N-末端结构域也调节催化活性，但在这种情况下，调节剂是Ca++/CaM。同样，关于三维结构或CaM结合如何解除酶抑制的机制一无所知。在本申请的第一部分中，我们提出通过X射线晶体学和核磁共振技术确定PDE 2A的调节GAF结构域的一级结构。我们还建议使用这些信息沿着从直接结合和诱变获得的信息来探索配体占用允许GAF结构域调节酶的催化活性的分子机制。在中试实验中，我们已经能够第一次建立大规模的细菌表达和纯化协议的cGMP结合GAF结构域的PDE 2。我们还制定了更小的，但足够的表达方案的PDE 1A和2A全酶从Sf 9细胞。更重要的是，最近，我们已经能够确定可重复地生产晶体的GAF域的条件，其结晶度约为3.0埃。 我们在这里建议，以更高的分辨率在存在和不存在的调节配体的情况下，这个GAF域的结构进行细化。由于迄今为止我们只能在GAF结构域配体cGMP存在下获得晶体，我们进一步提出通过NMR技术确定非配体GAF结构域的三维结构。在完成PDE 2A GAF结构域的初始结构之后，我们打算使用类似的方法将它们与其它含有PDE的GAF结构域PDE 5A的cGMP结合GAF结构域的结构进行比较，PDE 10A和PDE 11 A。在第二部分中，我们建议确定心脏Ca 2 +/CaM依赖性PDE 1A的催化结构域的晶体结构和分离的PDRE 1A的CaM结合/抑制结构域的溶液结构。这种酶主要在心脏的基质细胞中表达，被认为调节影响基质沉积的cAMP和cGMP库。该酶作为Ca 2+和环核苷酸途径之间的关键点。我们最近已经制定了一个分离和纯化程序的催化亚基的PDE 1A，并已获得小晶体，结晶约8埃。我们建议生产更好的晶体和细化这种PDE的结构。最后，我们建议通过一系列NMR结构测定和互补结合/功能实验来确定PDE 1A的抑制结构域与催化亚基相互作用的位点和机制。

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.