Ca2+ and Mg2+ Binding Properties of GCAP-1

Abstract

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca 2؉ in the light and inhibits it in the dark when Ca 2؉ concentrations rise. We present the first direct evidence that Mg 2؉ -bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP- In this study, we continue to develop a concept for the processes through which Ca 2ϩ sensor proteins, GCAPs, 2 regulate their target enzyme, retinal guanylyl cyclase (RetGC), in photosensitive neurons of the retina between light and dark. In darkadapted photoreceptors, cGMP keeps a fraction of cGMPgated channels open, thus allowing Na ϩ and Ca 2ϩ to enter the outer segment (1, 2). Because Ca 2ϩ is continuously removed from the outer segments by a light-independent Na ϩ /K GCAPs are recoverin-like neuronal calcium sensor proteins within the EF-hand superfamily (14, 15). Like other recoverinlike proteins, GCAPs have four EF-hand domains, but based on their amino acid sequence only three of them are capable of binding metal ion. Although the general model of regulation requires that Ca 2ϩ be either bound or released by GCAPs, little is known about the actual Ca 2ϩ binding properties of individual GCAPs under physiologically relevant conditions or the actual conformational change in GCAPs that constitutes switching them between the activator and the inhibitor forms. A number of previous studies aimed to identify conformational changes in GCAP-1 between its activator and inhibitor forms using limited proteolysis (16 -18), chemical modification of endogenous cysteine residues (17), EPR spectroscopy (17), and tryptophan fluorescence spectroscopy (18 -21) were based on the assumption that GCAPs are only Ca 2ϩ -binding proteins and that the metalfree GCAPs are the activators of RetGC-1. Our recent finding demonstrated that Mg 2ϩ is essential for adjusting the Ca 2ϩ sensitivity of RetGC regulation by GCAPs to the actual physiological range of free Ca 2ϩ concentrations and provided the first evidence that GCAPs are both Ca 2ϩ and Mg 2ϩ sensor proteins (5, 13, 22) thus raising the possibility that at physiological free Ca 2ϩ and Mg 2ϩ concentrations in the light at least one of the EF-hands in GCAP-1 is in a Mg 2ϩ -bound, rather than metalfree, state. Importantly binding of Mg 2ϩ changes the intensity of the intrinsic tryptophan fluorescence of GCAP-1, and that implies that a significant structural difference exists between the Mg 2ϩ -bound and the metal-free GCAP-1 (13). In the present study, we determined both Ca 2ϩ and Mg 2ϩ binding properties of GCAP-1 using two independent methods and a wide range of mutations introduced in its EF-hand domains. We found the following. (i) At the physiological concentrations of free Mg 2ϩ and Ca 2ϩ that exist in the vertebrate photoreceptors in the light (3,23,24), three EF-hands in GCAP-1 are predominantly occupied with Mg 2ϩ ions; hence the physiological RetGC-activat...

Activation and Inhibition of Photoreceptor Guanylyl Cyclase by Guanylyl Cyclase Activating Protein 1 (GCAP-1)

“…Free myristic acid was added from a concentrated ethanol solution to the suspension of bacterial cells to a final concentration of 100 g/ml 20 min prior to the induction with 0.5 mM isopropyl ␤-D-thiogalactopyranoside. Three hours after the induction, the bacterial pellet was harvested and the recombinant GCAPs were purified as described previously in detail (22). The concentration of GCAP-1 and its mutants was determined in 20 mM Na-phosphate buffer (pH 6.5) containing 6 M guanidine hydrochloride using extinction coefficients at 280 nm computed for the individual mutants from their amino acid composition (31) utilizing the ExPASY proteomic WWW server software from the Swiss Institute of Bioinformatics (32).…”

“…of RetGC-In our previous study we found that disabling all three metal-binding EF-hands in GCAP-1 by mutations that nonselectively hampered both Ca 2ϩ and Mg 2ϩ binding failed to produce a constitutive, Ca 2ϩ -insensitive activation of RetGC-1 (22). That was contrary to what one would expect if the apo form of the GCAP were the activator form and argued that the substitution of Ca 2ϩ by Mg 2ϩ , rather than merely loss of Ca 2ϩ , was required to make GCAP-1 undergo the inhibitor-to-activator transition in the light.…”

“…The N-terminal EF-hand domains in GCAP-1 and GCAP-2 lack some of the oxygen-containing groups required for coordinating the metal ion (17), but are instead required for their interaction with RetGC (18 -20). We previously found that in the conditions that mimic light-adapted or dark-adapted photoreceptors, three other EF-hands in GCAP-1 are predominantly filled by either Mg 2ϩ or Ca 2ϩ , respectively (21,22). There are multiple reports that substitutions of the first and last coordinating amino acids inactivate EF-hands in NCS proteins (17,(22)(23)(24)(25).…”

“…However, our recent study of metal-binding properties of GCAPs has also indicated that preventing Mg 2ϩ binding in GCAP-1 hampers stimulation of RetGC-1 (22). We concluded from the previous study (22) that, because cation binding was not only required for inhibition of RetGC, but was also critical for RetGC activation under physiological conditions, the general view on the functioning of GCAPs as metalbinding proteins in RetGC regulation needed to be revised (5)(6)(7)26). Therefore, the functions of the specific EF-hands in GCAPs must be revisited to provide more adequate understanding of their role in RetGC regulation via Ca 2ϩ /Mg 2ϩ exchange.…”

“…We previously found that in the conditions that mimic light-adapted or dark-adapted photoreceptors, three other EF-hands in GCAP-1 are predominantly filled by either Mg 2ϩ or Ca 2ϩ , respectively (21,22). There are multiple reports that substitutions of the first and last coordinating amino acids inactivate EF-hands in NCS proteins (17,(22)(23)(24)(25). Among these are the observations that inactivation of all three metal-binding EF-hands in GCAP-2 by substitution of the last Glu in EF-hands 2 and 3 with Gln and the first Asp in the EF-hand 4 with Asn make GCAP-2 a constitutive, insensitive to Ca 2ϩ activator of RetGC (24).…”