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

Abstract

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1−7). GCAPs form dimers that bind to Ca 2+ and confer Ca 2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg 2+ -bound, Ca 2+ -free, Fe 2+ -free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca 2+ -bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe 2+ binding. Nitroxide spinlabel probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe 2+ -dependent regulation of cyclase activity in zebrafish photoreceptors.

Structural basis of retinal membrane guanylate cyclase regulation by GCAP1 and RD3

“…GCAP1 and GCAP5 both form homodimers in solution in the absence of RetGC1 ( Figures 3C,D ). The dimerization of GCAP1 and GCAP5 are both Ca 2+ -independent and have a dimerization dissociation constant in the micromolar range ( Lim et al, 2018 ; Boni et al, 2020 ; Cudia et al, 2021 ). Structural models of GCAP1 and GCAP5 dimers were both determined previously by measuring intermolecular DEER distances that served as restraints for molecular docking ( Lim et al, 2018 ; Cudia et al, 2021 ; Figures 3C,D ).…”

“…The dimerization of GCAP1 and GCAP5 are both Ca 2+ -independent and have a dimerization dissociation constant in the micromolar range ( Lim et al, 2018 ; Boni et al, 2020 ; Cudia et al, 2021 ). Structural models of GCAP1 and GCAP5 dimers were both determined previously by measuring intermolecular DEER distances that served as restraints for molecular docking ( Lim et al, 2018 ; Cudia et al, 2021 ; Figures 3C,D ). The overall structures of both dimers are fairly similar (RMSD = 2.4 Å) and residues at the dimer interface are highly conserved (see red residues in Figure 3 ).…”

“…The overall structures of both dimers are fairly similar (RMSD = 2.4 Å) and residues at the dimer interface are highly conserved (see red residues in Figure 3 ). An important structural difference is that the GCAP5 dimer forms an intermolecular salt bridge between R22 and D71 that is not seen in the GCAP1 dimer, and the GCAP5 mutation R22A abolishes dimerization ( Cudia et al, 2021 ). The dimer structures of GCAP1 and GCAP5 are both stabilized by hydrophobic contacts at the dimer interface ( Figures 3C,D ).…”

“…The most prominent intermolecular contacts involve conserved hydrophobic residues, H19, Y22, M26, F73, V77, and W94 in GCAP1 ( Figure 3 ). In particular, the methyl side-chain atoms of V77 (V76 in GCAP5) each contact one another at the dimer interface and therefore explain why the V77E (or V76E in GCAP5) mutation significantly weakens protein dimerization for both GCAP1 and GCAP5 ( Lim et al, 2016 ; Cudia et al, 2021 ). Individual point mutations at the dimer interface in GCAP1 (H19A, Y22A, F73A, V77E, and W94A) or GCAP5 (H18A, Y21A, F72A, V76E, and W93E) each weaken the dimerization dissociation constant and completely abolish the activation of RetGC1 by GCAP1 ( Lim et al, 2018 ) or GCAP5 ( Cudia et al, 2021 ).…”

“…In this review, I provide an overview of previous atomic-resolution structures of GCAP1 ( Stephen et al, 2007 ; Lim et al, 2016 ), GCAP5 ( Cudia et al, 2021 ) and RD3 ( Peshenko et al, 2019 ), and propose a molecular mechanism for how RetGC1 might be regulated by GCAP1 and RD3.…”