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.

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca] and as an activator at low [Ca]. Recently, a novel missense mutation (G86R) was found in , the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the - and -domains of GCAP1, resulting in decreased Casensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg and Ca, moreover, substitutions of W94 abolished "phase II" in Ca-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca supported the presence of two cation-π interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions.