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Igor V Peshenko, Elena V Olshevskaya, Sunghyuk Lim, James Ames, Alexander M Dizhoor; The target enzyme-interfacing domain in photoreceptor guanylyl cyclase activating protein 1 (GCAP1). Invest. Ophthalmol. Vis. Sci. 2014;55(13):416.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal guanylyl cyclase (RetGC) activating proteins (GCAPs) regulate visual photoresponse and trigger various congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. In the present study, we describe a fine mapping of the residues in GCAP1 using global mutagenesis of the surface-exposed residues combined with the functional tests that allow distinguishing between the primary binding to the cyclase versus its activation.
Myristoylated GCAP1 mutants expressed in E.coli were purified and screened for their ability to activate RetGC1 in vitro in comparison with the wild type GCAP1. Fluorescently tagged GCAP1 mutants were co-expressed in HEK293 cells with fluorescently tagged RetGC1 and their association was tested in cyto using confocal microscopy.
In total, 107 residues, surface-exposed based on the Ca2+GCAP1 crystal structure , were altered, mostly using single point mutations. The side chains strongly affecting GCAP1 ability to activate RetGC1 localized to a distinct patch on the protein surface formed by non-metal binding EF hand 1, the loop and the exiting helix of a metal-binding EF-hand 2 and the entering helix of a metal binding EF-hand 3. Mutations in that region were able to completely block activation of the cyclase without affecting Ca2+ binding stoichiometry of GCAP1 or the overall backbone fold of the molecule. Most of the mutants that failed to activate RetGC1 also failed to co-localize with RetGC1 in HEK293 membranes. However, some GCAP1 mutants that completely failed to stimulate RetGC1 not only retained their ability to bind RetGC1 in cyto, but also markedly reduced RetGC1 activation by competing with the wild type GCAP1. These findings indicate that besides the primary binding RetGC1 activation by GCAP1 requires additional secondary interactions.
The residues required for GCAP1 binding to RetGC1 form a distinct “binding patch” on one side of the molecule and also contains at least two residues that are not essential for the primary binding but affect secondary interactions required for the RetGC1 activation. References: Stephen et al. (2007) Structure 15, 1392-1402
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