NMR STRUCTURAL STUDY ON PDE? IN COMPLEX WITH THE PDE5/6 CATALYTIC DOMAIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 脊椎动物视觉信号转导的中枢效应酶cGMP磷酸二酯酶（PDE 6）是一种abgg异源四聚体，含有两个同源催化亚基（a和B）和两个相同的抑制性g亚基（PDE g）。PDEg通过其C末端与ab催化二聚体的催化结构域结合而使PDE 6保持在失活状态。公认的PDE 6激活模型需要GTP激活的转导蛋白a亚基（GatGTP）与每个抑制性PDEg亚基结合，这从ab中的催化口袋中物理去除PDEg C末端，从而解除抑制性约束。虽然PDEg抑制口袋已被映射到ab催化结构域的生化研究，有没有任何原子结构可用于PDEg与ab催化亚基的相互作用。我们初步的NMR数据显示，在溶液中游离PDEg的C末端可能存在α螺旋。先前的诱变研究表明，PDEg的C末端在与ab催化结构域结合时是延伸的连续α螺旋; GatGTP与PDEg C末端位置76区域的相互作用触发C末端的构象变化并从催化口袋中置换PDEg。我们建议通过研究其与PDE 6催化结构域复合的NMR结构来解决有关PDEg在光转导中的作用的基本问题，利用我们建立的有效生产[U-13 C，15 N]标记PDEg的方法和最近在E.构建了PDE 5/6嵌合催化结构域（命名为hI 34）在大肠杆菌中的表达。该嵌合构建体使用PDE 5催化结构域作为模板产生，PDE 6 a （与PDE 6 a和B高度同源）催化口袋。将追求两个目标。1.）的人。研究与单独PDEg相比，PDEg C末端与hI 34构建体结合后是否发生构象变化。2.）的情况。阐明与hI 34复合物中PDEg C末端的结构是否与差距复合物中的结构不同，如晶体结构所示。从这项研究中获得的知识有望为PDEg抑制PDE 6和GatGTP激活PDE 6的机制提供清晰而重要的见解，这对于理解视觉转导至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central effector enzyme of vertebrate visual signal transduction, the cGMP phosphodiesterase (PDE6), is an abgg heterotetramer containing two homologous catalytic subunits (a and b) and two identical inhibitory g subunits (PDEg). PDEg keeps PDE6 at the inactivated state through its C terminus binding to the catalytic domain of the ab catalytic dimer. The accepted model for PDE6 activation requires binding of the GTP-activated transducin a subunit (GatGTP) to each inhibitory PDEg subunit, which physically removes the PDEg C terminus from the catalytic pocket in ab thus relieving the inhibitory constraint. Although the PDEg inhibition pocket has been mapped to the ab catalytic domain by biochemical studies, there is no any atomic structure available for interactions of PDEg with the ab catalytic subunits. Our preliminary NMR data show possible a helices in the C terminus of free PDEg in solution. Previous mutagenesis study suggests that the C terminus of PDEg is an extended continuous a helix when bound to the ab catalytic domain; interaction of GatGTP with the PDEg C-terminal position 76 region triggers a conformational change of the C-terminus and displaces PDEg from the catalytic pocket. We propose to address basic questions regarding the role of PDEg in phototransduction by studying its NMR structure in complex with the PDE6 catalytic domain, taking the advantage of our established method for efficient production of [U-13C,15N] labeled PDEg and the recent breakthrough in E. coli expression of the chimeric PDE5/6 catalytic domain (named hI34). This chimeric construct was generated using the PDE5 catalytic domain as a template into which the PDE6a (highly homologous to PDE6a and b) catalytic pocket was inserted. Two goals will be pursued. 1.) To investigate if a conformational change of the PDEg C terminus occurs upon its binding to the hI34 construct as compared to PDEg alone. 2.) To elucidate if the structure of the PDEg C terminus in a complex with hI34 is distinct from that in the GAP complex as revealed by the crystal structure. Knowledge gained from this study is expected to provide clear and important insights into the mechanisms of PDEg inhibition on PDE6 and PDE6 activation by GatGTP, which are critical for understanding of the visual transduction.