Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors

Abstract

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two non-conserved 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 analysis 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 both 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 a [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Fe2+ binding to GCAP5 decreases its ability to activate photoreceptor human GC-E by decreasing GC-activity more than 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.

Dimerization of Neuronal Calcium Sensor Proteins

“…The amino acid sequence of the zebrafish homolog called GCAP5 is the most divergent compared to the amino acid sequences of mammalian GCAP1 and GCAP2 (Figure 1 ). Two non-conserved Cys residues in GCAP5 (Cys 15 and Cys17) were shown recently to ligate Fe 2+ (Lim et al, 2017 ). Fe 2+ -binding to GCAP5 serves as a potent inhibitor and the Fe 2+ -bound GCAP5 is unable to activate RetGC at low Ca 2+ levels in light-adapted photoreceptors, suggesting that Fe 2+ binding to GCAP5 may serve as a redox sensor for phototransduction in zebrafish photoreceptors (Lim et al, 2017 ).…”

“…Two non-conserved Cys residues in GCAP5 (Cys 15 and Cys17) were shown recently to ligate Fe 2+ (Lim et al, 2017 ). Fe 2+ -binding to GCAP5 serves as a potent inhibitor and the Fe 2+ -bound GCAP5 is unable to activate RetGC at low Ca 2+ levels in light-adapted photoreceptors, suggesting that Fe 2+ binding to GCAP5 may serve as a redox sensor for phototransduction in zebrafish photoreceptors (Lim et al, 2017 ). Structurally, the Fe 2+ binding by Cys15 and Cys17 bridges two GCAP5 molecules into a [Fe(SCys) 4 ] dimeric complex (Lim et al, 2017 ) like that observed previously in two-iron superoxide reductases (deMaré et al, 1996 ; Min et al, 2001 ; Emerson et al, 2003 ).…”

“…The GCAP1-modulated RetGC1 transduction system also exists in the olfactory bulb (Duda et al, 2001 ). GCAP1, GCAP2 and GCAP5 each form a dimer in solution (Ermilov et al, 2001 ; Lim et al, 2017 , 2018 ) that binds to dimeric RetGC1 (Liu et al, 1997 ; Ramamurthy et al, 2001 ). The GCAPs activate RetGCs at low Ca 2+ levels in light activated photoreceptors (Peshenko and Dizhoor, 2006 ; Lim et al, 2009 ), whereas Ca 2+ -bound GCAPs inhibit RetGCs at high Ca 2+ levels in dark-adapted photoreceptors (Dizhoor and Hurley, 1996 ; Dizhoor et al, 1998 ).…”

“…In this review article, I discuss the recent atomic-resolution structures of dimeric forms of recoverin (Myers et al, 2013 ), GCAP1 (Lim et al, 2018 ), GCAP2 (Pettelkau et al, 2012 ), GCAP5 (Lim et al, 2017 ) and VILIP1 (Li et al, 2011 ) that each adopt very different quaternary structures (Figure 2 ). While the tertiary structures of each monomeric subunit are somewhat similar, the distinct quaternary structures and unique subunit packing arrangement at each dimer interface may play a role in facilitating target recognition and specificity.…”

Zebrafish Recoverin Isoforms Display Differences in Calcium Switch Mechanisms

“…The variety of zRec isoforms is reminiscent of zebrafish GCAPs, of which six isoforms are expressed in the zebrafish retina ( Imanishi et al, 2004 ; Rätscho et al, 2009 ). Detailed studies on zGCAP expression profiles in larval and adult animals, their regulatory properties, Ca 2+ sensitivities, and conformational dynamics revealed a differential action mode for each protein supporting a Ca 2+ relay mode of Ca 2+ -dependent negative feedback regulation ( Scholten and Koch, 2011 ; Fries et al, 2012 ; Sulmann et al, 2015 ; Lim et al, 2017 ). We suggest a similar regulatory mode for zRec forms.…”