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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脊椎动物视觉信号转导的中枢效应酶cGMP磷酸二酯酶(PDE6)是一种由两个同源催化亚基(a和b)和两个相同的抑制亚基(PDEg)组成的abgG异构体。PDEg通过其C端与ab催化二聚体的催化结构域结合，使PDE6处于失活状态。公认的PDE6激活模型需要将GTP激活的转导蛋白A亚单位(GatGTP)与每个抑制性PDEg亚基结合，这将物理上将PDEg C末端从ab中的催化口袋中移除，从而解除抑制限制。虽然生物化学研究已将PDEg抑制区定位于ab催化区域，但目前尚无PDEg与ab催化亚基相互作用的原子结构。我们的初步核磁共振数据表明，溶液中游离PDEg的C末端可能有螺旋结构。以往的诱变研究表明，PDEg的C末端是一个延伸的连续的螺旋，当结合到ab催化结构域时；GatGTP与PDEg C-末端第76位区域的相互作用会触发C-末端的构象变化，并取代催化口袋中的PDEg。我们建议利用我们建立的高效生产[U-13C，15N]标记PDEg的方法和最近在大肠杆菌中表达嵌合PDE5/6催化结构域(命名为hI34)的突破，通过研究PDEg与PDE6催化结构域的络合物的核磁共振结构来解决PDEg在光传导中的作用的基本问题。该嵌合构建体使用PDE5催化结构域作为模板，其中插入了PDE6a(与PDE6a和b高度同源)催化口袋。将追求两个目标。1)与单独使用PDEg相比，研究PDEg C末端在与hI34结构结合时是否发生构象变化。2.)为了阐明与hI34形成的复合体中PDEg C末端的结构是否与晶体结构所揭示的GAP复合体中的结构不同。这项研究的知识有望为PDEg抑制PDE6和GatGTP激活PDE6的机制提供明确和重要的见解，这对于理解视觉传导是至关重要的。

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.