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, 2024 May 21.
Article
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| MEDLINE
| ID: mdl-38662574
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 â¼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Modelos Moleculares
/
Proteínas Activadoras de la Guanilato-Ciclasa
/
Guanilato Ciclasa
Límite:
Animals
Idioma:
En
Revista:
Biochemistry
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos