Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation

Abstract

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Figure 1. Structural features of CaMKII and dimerization of the kinase domain.

A) Domain organization of CaMKII. The catalytic, regulatory and association domains are labelled, and predicted unstructured regions are shown in red. Sites of regulatory phosphorylation and oxidation are indicated. N- and C-terminal boundaries of the crystallized catalytic domain constructs are highlighted by boxed-in residues within the insets. The C-terminal boundary of CaMKIIδ, which is C-terminal to the range depicted (S333) has not been included in the figure. The organization of the autoinhibitory and the Ca²⁺/CaM binding domain is shown in the boxed alignment below the cartoon. For comparison, the sequence of the C. elegans (Ce) orthologue has also been included in the alignment. Secondary structural elements observed in the autoinhibited CaMKIIδ, CaMKIIδ/Ca²⁺/CaM, and C. elegans structure are shown above the alignment. The regions encompassing the Ca²⁺/CaM binding and autoinhibitory domains are highlighted in blue and green, respectively, and the phosphorylation sites are highlighted in yellow. B) Structural overview of the autoinhibited CaMKIIδ kinase domain refined at 2.3 Å resolution. The inhibitor bound at the ATP site is shown in surface representation in light-green, the activation segment is highlighted in magenta and main secondary structural elements are labelled. C) AUC sedimentation velocity experiment showing self-association of CaMKIIδ in absence (black line) and presence (red line) of a substrate-competitive peptide. The shift in sedimentation coefficient upon peptide binding is indicated by an arrow.

Figure 2. Structure of the CaMKIIδ/Ca²⁺/CaM complex.

A) Ribbon diagram of the autoinhibited CaMKIIδ kinase featuring the regulatory domain blocking the substrate binding site. Regulatory phosphorylation sites (T287, T306, T307) are marked by spheres. The region encompassing the autoinhibitory sequence and the Ca²⁺/CaM binding domain are colored green and blue respectively throughout. Helices are coloured in orange, beta sheets in light-blue, and the ATP-competitive inhibitor is shown in stick representation. B) Ribbon diagram of the CaMKIIδ/Ca²⁺/CaM structure showing the regulatory region interacting with Ca²⁺/CaM (colored in cyan) and a symmetry-related trans-phosphorylating ‘activating’ catalytic domain (colored in gray). The kinase domain is shown in the same orientation as in A. For clarity, the Ca²⁺/CaM associated with the trans-phosphorylating kinase has been omitted in the figure. The phosphorylation site T287 is labeled and highlighted with a sphere. C) Structural rearrangement of the autoregulatory domain upon binding of Ca²⁺/CaM. The inhibitory helix and the Ca²⁺/CaM binding motif are colored in green and blue, respectively, similar to the other subfigures D) Structural rearrangements in the catalytic domain upon binding of Ca²⁺/CaM, shown by superimposing the structures of the catalytic domain in its autoinhibited (light blue) and active (orange) state. The right panel shows an overview and on the left a detailed view of the observed conformational changes in the lower kinase lobe is shown. For clarity, the autoinhibitory helix (αI) and coil regions are rendered transparent and outlined in black. Residues discussed within the text are labeled.

Figure 3. Details of the substrate-peptide interaction.

A) Binding of the substrate sequence, including the regulatory site T287 (corresponding to T286 in the α isozyme), within the regulatory domain to the substrate binding pocket of an interacting catalytic domain. Substrate residues (green carbons) are labeled in bold and substrate positions are labeled with small Arabic numbers. The side chain of R284 forms hydrogen bonds with residues located in helix αD (E97/E100). Carbon atoms in catalytic domain residues are shown in light grey and residues are labeled in black. B) Electron density for the substrate peptide region. A region of the sigmaA-weighted electron density (2mFo-DFc) is shown contoured at 1σ superposed onto the final model. C) AUC velocity experiment showing CaMKIIδ/Ca²⁺/CaM association in the absence (black line) and presence (red line) of the substrate-competitive peptide encompassing the Ca²⁺/CaM binding region sequence. The assignment of the peaks to the oligomeric states (orders of association of CaMKII/Ca²⁺/CaM heterodimers) are indicated.

Figure 4. Recognition of the T307 autophosphorylation site and Ca²⁺/CaM binding.

A) Substrate interaction of T307 in autoinhibited CaMKIIδ. The coloring of the inhibitory helix and the Ca²⁺/CaM-binding domain is similar to Figure 2 and important residues discussed in the text are annotated. Regulatory phosphorylation sites are highlighted by a yellow star. B) Details of the interaction between the Ca²⁺/CaM binding site (blue helix) and Ca²⁺/CaM, shown as ribbon with semitransparent binding surfaces. Residues located in the interface are labeled. C) Isothermal titration calorimetry (ITC) showing binding of Ca²⁺/CaM to CaMKIIδ (left panel) and the isolated Ca²⁺/CaM binding domain (right panel). The figure shows raw injection heats (upper panel) and a binding isotherm of normalized integrated binding enthalpies (lower panel). Experiments were carried out in 20 mM HEPES pH 7.5, containing 150 mM NaCl, 5 mM DTT and 1 mM CaCl₂.

Figure 5. Oligomerization domains of human CaMKIIδ and CaMKIIγ.

A) Comparison between the structures of the dodecameric (CaMKIIγ) (upper) and tetradecameric (CaMKIIδ) (lower) arrangements. The rings are viewed looking face-on (left) and side-on (right) B) Model of the autoactivation of full-length CaMKII. For clarity, only one CaMKII dimer is highlighted (protomers shown in green and red, respectively). The model was constructed by manual docking of the kinase domain and the CaMKII/Ca²⁺/CaM complex to the experimental structure of the dodecameric oligomerization domain.

Figure 6. Putative mechanisms of CaMKII activation and inactivation.

In autoinhibited CaMKII (second from the left), the inactivating C-terminal helix is bound to the substrate binding site (shown as a red surface). Ca²⁺/CaM-independent mechanisms lead to an active state (“ON” state; to the top left) by methionine oxidation, or to an inactive state (“OFF” state; bottom left) by CASK-mediated T306/T307 phosphorylation. Ca²⁺/CaM-dependent activation (to the right) is achieved through structural rearrangement of the inhibitory helix caused by Ca²⁺/CaM binding and subsequent autophosphorylation of T287. Even when calmodulin is released from the complex following a drop in the Ca²⁺ level, re-binding of the inhibitory helix is blocked (marked as a red cross) due to pT287 and a structural change in the lower kinase lobe that closes the binding site for the helix, restructures the substrate binding site (depicted as a green surface), and aligns E97 in the active site for ATP binding. As a consequence, the active state of the kinase is long-lasting and signaling pathways that lead to changes in gene transcription are activated.