Retinal guanylyl cyclase activating protein 1 forms a functional dimer

Abstract

Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.

Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition

“…NCS proteins can form dimers, and the process may be dynamically influenced by the presence of cations [36]. Since it was recently proven that bovine GCAP1 acts as a functional dimer [37], we tested whether human GCAP1 variants showed similar oligomeric properties under GC activating/inhibiting conditions. To assess the quaternary structure of the GCAP1 variants we employed a combination of dynamic light scattering (DLS) and analytical size exclusion chromatography (SEC).…”

Dimerization of Neuronal Calcium Sensor Proteins

“…The GCAP1-modulated RetGC1 transduction system also exists in the olfactory bulb (Duda et al, 2001 ). GCAP1, GCAP2 and GCAP5 each form a dimer in solution (Ermilov et al, 2001 ; Lim et al, 2017 , 2018 ) that binds to dimeric RetGC1 (Liu et al, 1997 ; Ramamurthy et al, 2001 ). The GCAPs activate RetGCs at low Ca 2+ levels in light activated photoreceptors (Peshenko and Dizhoor, 2006 ; Lim et al, 2009 ), whereas Ca 2+ -bound GCAPs inhibit RetGCs at high Ca 2+ levels in dark-adapted photoreceptors (Dizhoor and Hurley, 1996 ; Dizhoor et al, 1998 ).…”

“…In this review article, I discuss the recent atomic-resolution structures of dimeric forms of recoverin (Myers et al, 2013 ), GCAP1 (Lim et al, 2018 ), GCAP2 (Pettelkau et al, 2012 ), GCAP5 (Lim et al, 2017 ) and VILIP1 (Li et al, 2011 ) that each adopt very different quaternary structures (Figure 2 ). While the tertiary structures of each monomeric subunit are somewhat similar, the distinct quaternary structures and unique subunit packing arrangement at each dimer interface may play a role in facilitating target recognition and specificity.…”

Evolution of the calcium feedback steps of vertebrate phototransduction

“…The molecular mechanism of GCAP activation at lowered Ca 2+ concentrations involves the binding of Mg 2+ [ 32 ] to EF-2 and EF-3, thereby inducing a conformational change; the role of EF4 is unclear. In contrast to the case for recoverin, the overall change in tertiary structure for GCAP1 is relatively small [ 33 ]; in particular, the myristoyl group in GCAP1 remains fully buried in both states [ 34 – 36 ]. GCAP2 is also myristoylated, but whereas the removal of the myristoyl group results in a sevenfold reduction in activity of GCAP1, it has little effect on GCAP2 [ 37 ].…”

“…Recent evidence has shown that GCAP1 forms a functional homodimer [ 33 ], suggesting a 2 : 2 stoichiometry of interaction with the GC homodimer. In vitro experiments with mammalian proteins have shown that GCAP1 and GCAP2 are able to activate the two GCs, GC-E and GC-F, with comparable efficacy.…”