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 Ca²⁺-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca²⁺] and as an activator at low [Ca²⁺]. Recently, a novel missense mutation (G86R) was found in GUCA1A, 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 N- and C-domains of GCAP1, resulting in decreased Ca^2+-sensitivity 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 Mg²⁺ and Ca²⁺, moreover, substitutions of W94 abolished "phase II" in Ca²⁺-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca²⁺-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca²⁺ 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.

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