Chemical shift assignments of retinal guanylyl cyclase activating protein 5 (GCAP5) with a mutation (R22A) that abolishes dimerization and enhances cyclase activation

Retinal membrane guanylyl cyclases (RetGCs) in vertebrate rod and cone photoreceptors are activated by a family of neuronal Ca2+ sensor proteins called guanylyl cyclase activating proteins (GCAP1-7). GCAP5 from zebrafish photoreceptors binds to RetGC and confers Ca2+/Fe2+-dependent regulation of RetGC enzymatic activity that promotes the recovery phase of visual phototransduction. We report NMR chemical shift assignments of GCAP5 with a R22A mutation (called GCAP5R22A) that abolishes protein dimerization and activates RetGC with 3-fold higher activity than that of wild type GCAP5 (BMRB No. 51,783).

RetGC to replenish cGMP levels in order to restore the dark state (Koch and Helten 2008;Koch and Stryer 1988). Mutations in GCAP1 that weaken Ca 2+ binding or otherwise alter Ca 2+ -sensitive activation of RetGC are genetically linked to retinal diseases (Jiang andBaehr 2010, Payne, Downes et al., 1998).
GCAP5 in zebrafish photoreceptors binds to both Ca 2+ and Fe 2+ (Lim et al. 2017). The Ca 2+ -free forms of GCAP1 (Peshenko and Dizhoor 2006) and GCAP5 (Lim et al. 2017) both activate RetGC activity in light-adapted photoreceptors, whereas the Ca 2+ -bound GCAP1 (Peshenko and Dizhoor 2007) and Fe 2+ -bound GCAP5 (Lim et al. 2017) both inhibit RetGC in dark-adapted photoreceptors. The NMR structure of GCAP5 (Cudia et al. 2021) revealed that GCAP5 forms a dimer in solution with key amino acid residues at the dimer interface (H18, Y21, R22, M25, F72, V76 and W93) that are important for cyclase activation. The GCAP5 mutations H18E, M25E and V76E each abolish GCAP5 dimerization and prevent activation of RetGC (Cudia et al. 2021). These results suggested that GCAP5 dimerization might be essential for RetGC activation (Ames 2021, 2022). However, this hypothesis was refuted by the discovery that the R22A mutation of GCAP5 not only abolishes GCAP5 dimerization but also causes a 300% increase in RetGC activation compared to that of wild type (Cudia et al. 2021). We hypothesize that the R22A mutation might somehow alter the structure of GCAP5 to abolish its dimerization and 1 3 increase its potency for activating RetGC. We report here NMR resonance assignments for the Ca 2+ -free activator and monomeric form of Ca 2+ -free GCAP5 with the R22A mutation (called GCAP5 R22A ) to understand how this mutation abolishes protein dimerization and causes a 300% increase of RetGC activity compared to that of wild type GCAP5.

Extent of assignments and data deposition
Representative NMR assignments are illustrated by twodimensional NMR spectra of Ca 2+ -free GCAP5 R22A ( 15 N-1 H HSQC, Fig. 1A-B and 13 C-1 H HSQC, Fig. 1C). The resonance assignments were determined by analyzing 3D triple resonance NMR spectra of 13 C/ 15 N-labeled GCAP5 R22A . The highly resolved NMR peaks with uniform intensities indicate a stable and folded structure. Amide resonances assigned to Q19, L33 and I70 exhibited noteworthy downfield shifts, perhaps because these residues are flanked by nearby aromatic rings (W20, F35 and F72 respectively) (Fig. 1A). The amide resonances assigned to G68 and G147 have downfield chemical shifts that are caused by a strong hydrogen bond between the backbone NH of G68 (EF2)/ G147 (EF4) with side chain carboxyl groups of D63 (EF2)/ D142 (EF4), respectively. These strong hydrogen bonds are stabilized by an open conformation for both EF2 and EF4. It is unusual for Ca 2+ -free EF-hands to occupy an open conformation that is typically only formed by Ca 2+ -bound EFhands (Ikura 1996, Yap, Ames et al., 1999. However, the NMR structure of wild type Ca 2+ -free GCAP5 revealed that the Ca 2+ -free structures of EF2, EF3 and EF4 each adopt a pre-formed open conformation (Cudia et al. 2021), which might explain why the GCAP proteins exhibit such high affinity Ca 2+ binding in the nanomolar range (Lim, Peshenko et al., 2009). Spectral assignments were obtained for more than 94% of the main chain 13 C resonances ( 13 Cα, 13 Cβ, and 13 CO), 97% of non-proline backbone amide resonances ( 1 HN, 15 N), and 87% of side chain resonances (Fig. 1C). The unassigned residues (A22, N46, E74, Y75, and I136) had weak HSQC peaks caused by exchange broadening that prevented their assignment. Complete chemical shift assignments ( 1 H, 15 N, 13 C) of Ca 2+ -free GCAP5 R22A have been deposited in the BioMagResBank (http:// www. bmrb. wisc. edu) under accession number 51,783.
The assigned amide chemical shifts of Ca 2+ -free GCAP5 R22A (BMRB 51,783) are compared to those of Ca 2+ -free GCAP5 wild type (BMRB 51,784) to help identify residues that are structurally affected by the R22A mutation (Fig. 3A). Not surprisingly, the GCAP5 residues in EF1 (Q19, W20, Y21 and K23) that are closest to R22A exhibit the largest chemical shift perturbation (Fig. 3A, B). In addition, C-terminal residues R176, I177 and V178 also exhibit detectably large chemical shift perturbations. In the wild type GCAP5 structure (Cudia et al. 2021), the side-chain methyl groups of I177 are in close proximity with the side chain indole group of W20, and both side chains make close contact with the N-terminal myristoyl group (Fig. 3C). Interestingly, the myristoyl group contacts with both W20 and I177 are both important for the proposed Ca 2+ -myristoyl tug mechanism that transmits Ca 2+ -induced conformational changes from the EF-hands to the myristoyl group (Peshenko, Olshevskaya et al., 2012). We suggest that the R22A mutation may stabilize the Ca 2+ -free GCAP5 activator conformation by disrupting the Ca 2+ -myristoyl tug (Peshenko et al. 2012). The NMR assignments of Ca 2+ -free GCAP5 R22A presented here suggest the R22A mutation affects the structure in both EF1 (W20) and Funding Work supported by NIH grants to J.B.A (R01 EY012347) and to the UC Davis NMR Facility (RR11973).

Data availability
The assignments have been deposited to the BMRB under the accession code: 51,783.

Conflict of interest
The authors declare they have no competing conflict of interest.

Ethical approval
The experiments comply with the current laws of the United States.
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Fig. 3
Chemical shift perturbation (CSP) for Ca 2+ -free GCAP5 R22A versus wild type GCAP5. A Backbone amide CSP was calculated as: CSP = √ H N 2 + (0.14 × N) 2 . ΔH N and ΔN are the observed difference in the 1 H N and 15 N chemical shifts, respectively between Ca 2+ -free GCAP5 R22A (BMRB 51,783) and wild type GCAP5 (BMRB 51,783). B CSPs mapped on the structure of GCAP5 (Cudia et al. 2021). C Close-up view of the myristoyl group binding site environment in GCAP5. The side chains of W20 and I177 make close contacts with the myristoyl group