PUBLICATION
Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein-5 from Zebrafish Photoreceptors
- Authors
- Lim, S., Scholten, A., Manchala, G., Cudia, D., Zlomke-Sell, S.K., Koch, K.W., Ames, J.B.
- ID
- ZDB-PUB-171128-1
- Date
- 2017
- Source
- Biochemistry 56(51): 6652-6661 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Binding Sites
- Ferrous Compounds/metabolism*
- Guanylate Cyclase/antagonists & inhibitors
- Guanylate Cyclase/metabolism*
- Guanylate Cyclase-Activating Proteins/chemistry
- Guanylate Cyclase-Activating Proteins/genetics
- Guanylate Cyclase-Activating Proteins/metabolism*
- Light
- Mutation
- Nuclear Magnetic Resonance, Biomolecular
- Photoreceptor Cells, Vertebrate/metabolism*
- Protein Binding
- Protein Multimerization
- Retina/metabolism*
- Sequence Homology, Amino Acid
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism*
- PubMed
- 29172459 Full text @ Biochemistry
Citation
Lim, S., Scholten, A., Manchala, G., Cudia, D., Zlomke-Sell, S.K., Koch, K.W., Ames, J.B. (2017) Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein-5 from Zebrafish Photoreceptors. Biochemistry. 56(51):6652-6661.
Abstract
Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn2+ and Fe2+). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe2+, suggesting that Fe2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe2+-free and Fe2+-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Binding of Fe2+ to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe2+-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping