Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid

Abstract

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKII^L308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

Keywords: UV; calcium; crosslinking; kinase.

FIGURE 1

Characterization of CaMKII^L308BzF. (a) CaMKII domain arrangement showing N‐terminal kinase domain (gray), followed by a regulatory domain (orange), a linker and a C‐terminal association or hub domain (blue). Phosphorylation residues are colored in yellow (T286 and T305/306, CaMKIIα numbering). Calmodulin recognition element is highlighted in red, and substitution of Leu at position 308 with BzF colored in purple. CaMKII holoenzyme is adapted using PDB:5U6Y. Cartoon representation of the CaMKII dodecamer with L308BzF residues colored purple and represented in spheres. (b) Coomassie‐stained SDS‐polyacrylamide gel showing rescue of CaMKII^L308TAG expression by growing E. coli in BzF‐containing medium. Uninduced—uninduced culture; WCL—whole cell lysate; CCL—cleared cell lysate; 15 mL beads—sample from Co²⁺ beads resuspended in 15 mL of wash buffer, 1 mL beads—same volume of sample from 1 mL Co²⁺ beads resuspended in 1 mL of wash buffer; CaMKII FL—full‐length CaMKII; λPP—Lambda Protein Phosphatase. (c) Chromatogram showing absorbance at 280 nm (mAU) from Superose 6 10/300 size exclusion run of purified CaMKII^L308BzF. An equivalent run for CaMKII^WT is shown for comparison. The first peak with elution volume (V _e) of 13.5 mL corresponds to dodecameric CaMKII holoenzyme (around 650 kDa). The second two peaks (V _e = 17.2 and 18.2 mL) correspond to smaller fragments of CaMKII. SDS gel shows the full length and fragments in different fractions from CaMKII^L308BzF run, as well as the sample loaded on the column (“load”). (d) Activity of CaMKII^WT (green line) and CaMKII^L308BzF (blue line) against CaMKII peptide‐substrate (Syntide), measured using ADPQuest kinase assay, showing comparable t_1/2 for the two proteins. Red line‐background signal, light green—CaMKII^WT activity in the absence of Ca²⁺/CaM, light blue—CaMKII^L308BzF activity in the absence of Ca²⁺/CaM. RFU—raw fluorescent units. The shading represents standard deviation (SD) from the mean value of three technical replicates.

FIGURE 2

UV‐induced crosslinking of Calmodulin to CaMKII^L308BzF is Ca²⁺‐dependent. (a) Schematic representation of the experiment done in (b). (b) Coomassie‐stained SDS‐polyacrylamide gel showing absence of UV‐induced crosslinking of Calmodulin to CaMKII^WT (first 6 lanes), and dependence of UV‐induced crosslinking of Calmodulin to CaMKII^L308BzF on Ca²⁺ (second 6 lanes). CaM—Calmodulin. (c) Coomassie‐stained SDS‐polyacrylamide gel showing UV‐induced CaMKII dimer formation when 1 mM TCEP is used in the CaMKII buffer (lanes 1, 2, 5, and 6) and absence of this band when the CaMKII buffer is supplemented with 50 mM TCEP in the case of CaMKII^WT (lanes 3 and 4), but not in the case of CaMKII^L308BzF (lane 8). His‐tagged Calmodulin (His‐CaM) is present in lanes 5 and 6.

FIGURE 3

Determination of Calmodulin binding constant. (a) Coomassie‐stained SDS‐polyacrylamide gel of samples containing CaMKII^L308BzF and varying concentrations of His‐Calmodulin (0.12–16 μM) supplemented with 2 mM CaCl₂, used to quantify UV‐dependent CaMKII^L308BzF monomer band depletion, used to construct the binding curve in panel (b). CaM—Calmodulin. (b) Calculation of Calmodulin dissociation constant (k _d), by fitting the densitometry of Calmodulin and UV‐dependent depletion of monomeric CaMKII^L308BzF bands from the gels in panel (a).

FIGURE 4

UV‐induced crosslinking of Calmodulin to CaMKII^L308BzF does not change the oligomeric state of CaMKII. (a) Mass distribution of 400 nM CaMKII^WT under resting conditions. The distribution consists of 7161 particle mass measurements (total counts). (b) Mass distribution of 400 nM CaMKII^L308BzF under resting conditions (6138 total counts). (c) Mass distribution of 400 nM CaMKII^WT incubated with Ca²⁺:Calmodulin (20,440 total counts). (d) Mass distribution of 400 nM CaMKII^L308BzF incubated with Ca²⁺:Calmodulin (8154 total counts). (e) Mass distribution of 400 nM CaMKII^WT incubated with Ca²⁺:Calmodulin and UV‐treated (24,153 total counts). (f) Mass distribution of 400 nM CaMKII^L308BzF incubated with Ca²⁺:Calmodulin, and UV‐treated (4964 total counts). Graph top panels—residuals of the Gaussian fit (red); middle panels—histogram of the counts (red line) with fitted Gaussian curve (dashed blue line for CaMKII^WT, dashed magenta line for CaMKII^L308BzF), bottom panels—Gaussian fits of the counts (blue line—CaMKII^WT, magenta line—CaMKII^L308BzF). The centers and widths of the fitted peaks are indicated.

FIGURE 5

Ca²⁺ is dispensable for the activity of constitutively Calmodulin‐bound CaMKII. (a) Schematic representation of experiments done in (b). (b) Western blot detection of the ability of CaMKII^WT (first 6 lanes) or CaMKII^L308BzF (second 8 lanes) to trans‐autophosphorylate itself under different conditions. Lane 1—CaMKII^WT activated with Ca²⁺:CaM, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 2—CaMKII^WT activated with Ca²⁺:CaM, and then incubated with SEC buffer instead of Mg²⁺:ATP, for 10 min at 37°C; lane 3—CaMKII^WT activated with Ca²⁺:CaM, followed by incubation with 7 mM EGTA, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 4—CaMKII^WT activated with Ca²⁺:CaM, followed by incubation with 40 mM EGTA, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 5—CaMKII^WT incubated with Ca²⁺:CaM, which was treated with 7 mM EGTA prior to incubation with CaMKII, followed by incubation with Mg²⁺:ATP for 10 min at 37°C; lane 6—CaMKII^WT incubated with Ca²⁺:CaM, which was treated with 40 mM EGTA prior to incubation with CaMKII, followed by incubation with Mg²⁺:ATP for 10 min at 37°C; M—marker; lane 7—CaMKII^L308BzF activated with Ca²⁺:CaM, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 8—CaMKII^L308BzF activated with Ca²⁺:CaM, and then incubated with SEC buffer instead of Mg²⁺:ATP, for 10 min at 37°C; lane 9—CaMKII^L308BzF activated with Ca²⁺:CaM, followed by UV treatment, and then incubated with Mg²⁺:ATP for 10 min at 37°C; M—marker; lane 10—CaMKII^L308BzF activated with Ca²⁺:CaM, incubated with 40 mM EGTA, UV treated, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 11—CaMKII^L308BzF activated with Ca²⁺:CaM, incubated with 40 mM EGTA, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 12—CaMKII^L308BzF activated with Ca²⁺:CaM, UV treated, then incubated with 40 mM EGTA, and then incubated with Mg²⁺:ATP for 10 min at 37°C; lane 13—CaMKII^L308BzF incubated with Ca²⁺:CaM, which was treated with 7 mM EGTA prior to incubation with CaMKII, followed by incubation with Mg²⁺:ATP for 10 min at 37°C, and then UV treatment; lane 14—CaMKII^L308BzF incubated with Ca²⁺:CaM, which was treated with 40 mM EGTA prior to incubation with CaMKII, followed by incubation with Mg²⁺:ATP for 10 min at 37°C, and then UV treatment. Blue box indicates that UV treatment was preceding the EGTA treatment; orange box indicates that incubation of Ca²⁺:CaM with EGTA was preceding the incubation with CaMKII; purple box indicates that EGTA treatment and activation preceded UV treatment. Arrowheads on the blots represent pT286 signal in respective lanes. Green bands on the blot represent fluorescence of the secondary antibody recognizing pT286, while the red bands on the blot represent fluorescence of the secondary antibody recognizing total CaMKII protein.