Abstract
Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca(2+) binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94.
Highlights
GCAP1 regulates cGMP synthesis in photoreceptors in response to light
We found that the residues required for GCAP1 binding to RetGC1 formed a distinct “binding patch” on one side of the molecule that contains at least two residues, Met-26 and Trp-94, that are not essential for the primary binding but affect secondary interactions required for RetGC1 activation
Mutagenesis and the Primary Screening for the Residues Affecting RetGC1 Activation—A total of 107 residues, surfaceexposed based on the Ca2ϩGCAP1 crystal structure [24], were altered, mostly using a single point mutation, in some cases residues were substituted in pairs or in larger blocks as indicated in Fig. 1B and Table 1
Summary
GCAP1 regulates cGMP synthesis in photoreceptors in response to light. Results: Mutagenesis of the entire GCAP1 surface reveals its guanylyl cyclase interface. Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. Most of the GCAP1 mutations introduced into the “binding patch” prevented co-localization with RetGC1, except for Met, Lys-85, and Trp-94 With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Of Basic Sciences and Pennsylvania College of Optometry, Salus University, 8360 Old York Rd., Elkins Park, PA 19027. Target-binding Interface on GCAP1 in the photoreceptor outer segment, the molecular mechanism of RetGC activation by GCAP remains obscure. There have been several attempts to identify the possible sites of target recognition in GCAPs using chimeras with other neuronal calcium sensor (NCS) proteins [32, 47, 49], implicating several regions in GCAP primary structure as likely parts of the cyclase-binding interface. We found that the residues required for GCAP1 binding to RetGC1 formed a distinct “binding patch” on one side of the molecule that contains at least two residues, Met-26 and Trp-94, that are not essential for the primary binding but affect secondary interactions required for RetGC1 activation
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