GUANYLATE CYCLASE ACTIVATING PROTEIN 1 MONOMER-DIMER EQUILIBRIUM CONTROLLED BY CA2+ OR MG2+ BINDING: HINTS TO UNDERSTAND RETINAL GUANYLATE CYCLASE REGULATION

Neuronal calcium sensors play a crucial role in different pathways of Ca2+-mediated neurotransmission. Among them guanylate cyclase-activating protein 1 (GCAP1) is expressed only in photoreceptors and activates or inhibits retinal guanylate cyclase 1 (retGC1) depending on cellular Ca2+ concentrations during phototransduction. To date, 22 pathogenic mutations responsible for retinal dystrophy have been associated to GCAP1, but a complete picture of the molecular determinants of the disease is still missing. The only crystal structure available so far is the wt Ca2+-bound monomeric homologue from chicken and no cure exists for retinal dystrophy. In this work I report for the first time that the recombinant human GCAP1 is characterized by a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and by the nature of the divalent ion bound. Surprisingly, I discovered that also the chicken protein shows a similar mechanism, suggesting that this property could be potentially functional for GCAP1 activity and conserved among different species. Despite the large number of crystallization trials, no diffracting crystal of the human GCAP1 was obtained, probably due to the flexible C-terminal tail and the intrinsic dynamicity of the protein. To overcome this issue, I produced a construct lacking the 12 C-term residues and stabilized by a disulfide bridge between the N- and C-term domains which was successfully crystallized. We showed that such engineered construct is able to regulate retGC1 as well as the wt protein. By combining SAXS, protein-protein docking and molecular dynamics simulation we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer which are stabilized by some of the residues involved in the interaction with the retGC1. We used a biophysical and biochemical approach to thoroughly investigate three pathogenic variants (D100G, E155A and E155G) characterized by mutations in residues directly involved in Ca2+-coordination. All the three variants were able to form oligomers in solutions, showing a decreased affinity for Ca2+ and constitutively activating retGC1 at physiological calcium concentrations. Besides local structural effects, the mutations perturb also the oligomeric state of GCAP1 suggesting that the multimeric assembly of the protein could affect its proper biological function. A recombinant baculovirus for the expression of the cytoplasmic domain of retGC1 in insect cells was produced with the aim to get atomic structural information on the GCAP1/retGC1 complex. This will facilitate the identification of drug candidates able to recognize the binding region of the pathogenic GCAP1 mutants with the cyclase and to competitively inhibit the constitutive retGC1 activation, restoring the homeostasis of second messengers which is impaired in retinal degenerative diseases. A preliminary molecular docking based on the crystal structure of the chicken protein was performed and I identified four molecules able to bind the wt human GCAP1 in the millimolar/micromolar range. Together these results shed new light on the quaternary assembly of the wt human GCAP1, showing how the structural changes related to the presence of Ca2+ or Mg2+ are reflected in the different measured dimerization constant. Such conformational changes are in turn likely related to the regulatory mechanism of GCAP1 in the modulation of the retGC1 activity. The differences between the oligomerization state of D100G, E155G and E155A variants suggest a correlation between the altered quaternary assembly of GCAP1 and the aberrant activity of the mutants, representing a step forward to dissect the structural bases of the altered regulatory mechanism of GCAP1 in retinal dystrophies.