Abstract
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 Ca2+-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.
Highlights
The phototransduction cascade is the first of a complex series of biochemical networks that mediate the conversion of a visual stimulus into a neuronal one
G86R+W94L variant greatly affected the maximal activation of the target, resulting in a three-and-a-half-fold decrease of synthesized cyclic guanosine monophosphate (cGMP) in the absence of Ca2+ with respect to the WT (20.4 vs. 5.8 nmol cGMP*min−1*mg protein−1, two-tailed t-test, n = 3, p-value = 0.006)
Both analytical size exclusion chromatography (SEC) and dynamic light scattering (DLS) results pointed towards an aggregation process of the Guanylate Cyclase (GC) inhibitor state of the pathological Guanylate Cyclase activating protein 1 (GCAP1) variant mediated by the concomitant presence of the G86R mutation and of aromatic residues in position 94
Summary
The phototransduction cascade is the first of a complex series of biochemical networks that mediate the conversion of a visual stimulus into a neuronal one. Some disease-associated mutations resulted in an unaltered Ca2+-sensitivity [24,33], suggesting that GC dysregulation may arise from higher affinity for Mg2+ or from alterations in the long-range allosteric communication between the EF-hands and the target interface [10] In this respect, the molecular characterization of the G86R GCAP1 mutant showed features in between the two groups, as the constitutive activation of the GC was due to a concerted effect of: (i) increased affinity for Mg2+; (ii) increased affinity for the target; and (iii) lower Ca2+-sensitivity. Such intramolecular lock prevents the physiological activator-to-inhibitor structural transition of G86R GCAP1 that would be expected at physiological levels of Ca2+ and is thought to be responsible for the constitutive activation of the GC target under conditions associated with retinal dystrophy
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