Biochemical and structural characterization of NORPA/INAD interaction

Abstract

Drosophila phototransduction is the fastest G-protein coupled signaling pathway known to date. Several key proteins including TRP, NORPA and ePKC, are assembled into a large complex by a master scaffold protein, INAD, which contains five PDZ domains. Previous study in our lab discovered that INAD PDZ2 binds to eye PKC and INAD PDZ3 binds to TRP. However, the binding partners of INAD PDZ5 and the molecular mechanism governing their interactions are unknown. Here, we discovered that NORPA CTD binds to INAD PDZ45 strongly with a dissociation constant of 20 nM, more than 100-fold stronger than canonical PDZ-PBM interactions. Apo-form NORPA crystal structures, combined with NMR and ITC data, reveal a supramodule binding model for NORPA/INAD interaction. In addition, we discovered that NORPA CTD undergoes pH-dependent changes of structural stability and membrane binding, which may represent another evidence for proton as a general second messenger in PLC/PIP₂ signaling processes.

Date of Award	2015
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology