Chemical shift assignments of retinal guanylyl cyclase activating protein 5 (GCAP5)

Abstract

Retinal membrane guanylyl cyclase (RetGC) in photoreceptor rod and cone cells is regulated by a family of guanylyl cyclase activating proteins (GCAP1-7). GCAP5 is expressed in zebrafish photoreceptors and promotes Ca 2+-dependent regulation of RetGC enzymatic activity that regulates visual phototransduction. We report NMR chemical shift assignments of the Ca 2+-free activator form of GCAP5 (BMRB no. 27705).

NMR and EPR-DEER Structure of a Dimeric Guanylate Cyclase Activator Protein-5 from Zebrafish Photoreceptors

“…The GCAP5 main chain structure (Figure B) contains a total of 11 α-helices and four β-strands: α1 (residues 8–15), α2 (19–27), α3 (35–41), α4 (50–62), α5 (72–82), α6 (88–97), α7 (108–121), α8 (130–140), α9 (151–160), α10 (162–172), α11 (175–182), β1 (32–34), β2 (69–71), β3 (105–107), and β4 (148–150). The secondary structure of the final NMR structure of Mg 2+ -bound GCAP5 (Figure B) is slightly different from the secondary structure inferred from the chemical shift index . In particular, the C-terminal helix (α11) defined by NOESY and RDC data was predicted to be a random coil based on chemical shift analysis .…”

“…The NMR structure of GCAP5 contains Mg 2+ bound at EF2 (orange sphere in Figure ) as evidenced by characteristic Mg 2+ -dependent amide chemical shift changes assigned to Gly68 in EF2 . Also, Mg 2+ -induced NMR spectral changes for GCAP5 (Figure S1) further demonstrate binding of Mg 2+ to GCAP5.…”

“…42,43 ■ RESULTS NMR-Derived Structures of Mg 2+ -Bound GCAP5. NMR chemical shift assignments published previously for Mg 2+ -bound GCAP5 44 were used in this study to determine NOESY-based distances, NMR-derived dihedral angle restraints, and residual dipolar coupling (RDC) restraints (Figure 2) that served as input for restrained molecular dynamics structure calculations (see Materials and Methods). The residual dipolar coupling magnitude and rhombicity were calculated by fitting the measured residual dipolar couplings to the calculated structure using the PALES program.…”

“…The GCAP5 main chain structure (Figure 3B The secondary structure of the final NMR structure of Mg 2+bound GCAP5 (Figure 3B) is slightly different from the secondary structure inferred from the chemical shift index. 44 In particular, the C-terminal helix (α11) defined by NOESY and RDC data was predicted to be a random coil based on chemical shift analysis. 44 The secondary structure elements of GCAP5 combine to form two separate domains: The Nterminal domain is formed by the N-terminal helix (α1), EF1 (green, residues 19−41), and EF2 (red, residues 50−82), and the C-terminal domain is formed by EF3 (cyan, residues 88− 121), EF4 (yellow, residues 130−160), and C-terminal helices (α10 and α11 in Figure 3B).…”

“…44 In particular, the C-terminal helix (α11) defined by NOESY and RDC data was predicted to be a random coil based on chemical shift analysis. 44 The secondary structure elements of GCAP5 combine to form two separate domains: The Nterminal domain is formed by the N-terminal helix (α1), EF1 (green, residues 19−41), and EF2 (red, residues 50−82), and the C-terminal domain is formed by EF3 (cyan, residues 88− 121), EF4 (yellow, residues 130−160), and C-terminal helices (α10 and α11 in Figure 3B). The N-terminal helix (α1) of GCAP5 is extended by two residues compared to that of GCAP1 (Figure 1).…”