Abstract
Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells.
Highlights
The development of effective fluorescent Ca2+ indicator proteins (FCIPs) is a challenge for a number of laboratories working with fluorescent proteins (FPs)
Earlier we described circularly permuted GFP-based Ca2+ sensors Case12 and Case16 with superior dynamic ranges of up to 12-fold and 16.5-fold increase in green fluorescence between
The overall structure of these sensors is a monomer consisting of circularly permuted green fluorescent protein (cpGFP) “core” in its typical β-barrel shape inserted between an M13-peptide and CaM-domains
Summary
The development of effective fluorescent Ca2+ indicator proteins (FCIPs) is a challenge for a number of laboratories working with fluorescent proteins (FPs). We report the crystal structure of the high-contrast GCaMP-like (see Supplementary Figure 1 for protein sequence alignment) Ca2+ sensor Case16 [7] in the presence of low Ca2+ concentration. At this intermediate stage of Ca2+-dependent response Case is characterized by incomplete interaction of CaM with its target M13-peptide and its chromophore environment differs significantly from that of GCaMP2 in its Ca2+-saturated form reported earlier [14,15]. Our data contribute to the structural understanding of cpGFP-based Ca2+ sensors and could enable the development of improved genetically encoded sensor variants for Ca2+ and other analytes
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