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Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors
journal contribution
posted on 2017-11-27, 00:00 authored by Sunghyuk Lim, Alexander Scholten, Grace Manchala, Diana Cudia, Sarah-Karina Zlomke-Sell, Karl-W. Koch, James B. AmesSensory
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.
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Keywords
GCAP 5GCAP 5 dimerthiol side chainsRetinal Guanylate Cyclase Activator Protein 5guanylate cyclase activator proteinsCys 15nonconserved cysteine residuestransition state metal ionsGCAP 5 formstwo-iron superoxide reductasesGC-ECys 17. Size exclusion chromatography analysisNMRFerrous Ion BindingITCZebrafish Photoreceptors Sensory guanylate cyclasesGCAP 5 molecules15A
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