PUBLICATION
NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation
- Authors
- Cudia, D.L., Ahoulou, E.O., Bej, A., Janssen, A.N., Scholten, A., Koch, K.W., Ames, J.B.
- ID
- ZDB-PUB-240426-4
- Date
- 2024
- Source
- Biochemistry 63(10): 1246-1256 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Animals
- Calcium/metabolism
- Enzyme Activation
- Guanylate Cyclase*/chemistry
- Guanylate Cyclase*/genetics
- Guanylate Cyclase*/metabolism
- Guanylate Cyclase-Activating Proteins*/chemistry
- Guanylate Cyclase-Activating Proteins*/genetics
- Guanylate Cyclase-Activating Proteins*/metabolism
- Models, Molecular
- Mutation
- Nuclear Magnetic Resonance, Biomolecular
- Protein Conformation
- Protein Multimerization
- Retina/metabolism
- Zebrafish*/metabolism
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 38662574 Full text @ Biochemistry
Citation
Cudia, D.L., Ahoulou, E.O., Bej, A., Janssen, A.N., Scholten, A., Koch, K.W., Ames, J.B. (2024) NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation. Biochemistry. 63(10):1246-1256.
Abstract
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping