Conformational changes underlying calcium/calmodulin-dependent protein kinase II activation

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.

Figure 1

(A) Schematic representation of the full-length CaMKII gene and the monomer construct used for these studies. The regulatory domain is highlighted in red with the CaM-binding region in orange. The R1, R2, and R3 refer to segments of the regulatory domain defined previously (Chao et al, 2010). (B) Far-UV and (C) near-UV circular dichroism analysis for the wild-type and cysteine-less CaMKII (WT^*) along with their D135N inactivated counterparts. The spectra are almost superimposable confirming the lack of substantial structural changes due to the cysteine substitutions. (D) Stability curves demonstrating the lack of substantial effects on melting temperature by the cysteine substitutions.

Figure 2

(A) Analysis of spin label dynamics at site 307. The experimental EPR spectrum was fit by two components using the MOMD analysis as described in the Materials and methods section. The fast component (green spectrum) arises from an unstructured, undocked conformation of the R3 segment while the slow (orange spectrum) component arises from a docked conformation. (B) Percentages of the fast and the slow components for representative residues in the R3 segment in the apo CaMKII state (black bars) and the ATP-bound state (purple bars). (C) Inverse correlation times (τ⁻¹) for sites in the R1 region plotted versus residue number. The correlation time was determined from non-linear least-squares analysis of the EPR spectra (see Materials and methods). The superimposed sinusoid fit to the data has a 3.7 period. The local environment of these residues in the crystal structure (PDB 2BDW) is shown to highlight the agreement with the EPR data (image created with PyMOL, DeLano Scientific (DeLano, 2002)). Thus, residues on the surface (green) (e.g. 289) have a large τ⁻¹ while those at the interface with the catalytic domain (orange) have small τ⁻¹.

Figure 3

Representative distance distributions between (i, i+4) spin label pairs in the R1; 286/290 (A) and R3; 300/304 (B) segments. The distributions, consisting of the probability of given a distance P(r), were obtained from fits to the EPR spectra as detailed in the Materials and methods and shown in Supplementary Figures S5 and S7A. Ca²⁺/CaM binding to R3 induces conformational changes along the entire regulatory segment evidenced by changes in average distance and breadth of the distribution (A, red trace) and increased dynamics of the R1 segment illustrated by sharper EPR lineshapes at sites 287 and 282 (C) and increases in the inverse correlation times (D).

Figure 4

(A) Comparison of the inverse rotational correlation time of spin labels in three CaMKII intermediates showing the increase in dynamics of the R1 and R2 segments in the presence of Ca²⁺/CaM and in the T286E mutant. (B) The phosphorylation-mimicking mutation T286E destabilizes the R1 helix leading to an increase in the width of the distance distribution between (i, i+4) spin label pairs. (C) R1 helix unfolding is manifested by a shift in the melting temperature of T286E. (D) Representative EPR lineshapes highlighting the increased dynamics of the regulatory domain in the CaM-bound T286E mutant.

Figure 5

(A) Model of the mechanism of autoinhibition and activation of CaMKII. The CaMKII monomer is represented with N- and C-lobes of the catalytic domain in blue and the regulatory domain in red. The ATP-binding and Thr286-docking sites are indicated in grey with letters ‘A’ and ‘T’, respectively. In the basal, apo intermediate, the R3 segment of the regulatory domain is in a dynamic equilibrium between docked and undocked, flexible (indicated by parentheses) conformations. ATP binding shifts the equilibrium towards the undocked conformation facilitating exposure of the catalytic cleft. CaM (yellow) binding releases autoinhibition and primes the regulatory domain for phosphorylation at residue 286. Autophosphorylation at Thr286 causes further flexibility of the regulatory domain and exposes site 293, which is implicated in CaM trapping. While CaM dissociation allows for reinstatement of the R3 equilibrium, the R1 helix remains predominantly unstructured. (B) Cartoon model of cooperative activation in the holoenzyme. CaM binding to one subunit causes undocking and unfolding of R1. The released R1 can displace the R3 segment of a neighbouring kinase subunit from the catalytic cleft. This R3 is undocked and primed for CaM association.

Figure 6

Shifts in the R3 equilibrium induced by binding of autocamtide-2 (AC-2). EPR spectra for site 307 show that the level of the mobile spectral component arising from undocked conformation of the R3 segment increases in the presence of AC-2. The T286E mutation increases the affinity of this interaction as evidenced by the lower molar ratio of peptide to CaMKII (x axis) required to increase the mobile spin label fraction.