This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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University of Wisconsin Madison
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