A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation

Abstract

Memory formation, fertilization, and cardiac function rely on precise Ca²⁺ signaling and subsequent Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Ca²⁺ sensitivity of the four CaMKII paralogs in mammals is linked to the length of the variable linker region that undergoes extensive alternative splicing. In this study, we determine that the position of charged residues within the linker modulates the Ca²⁺/CaM sensitivity. We present an X-ray crystal structure of the full-length CaMKIIδ holoenzyme consisting of domain-swapped dimers within a dodecameric complex, revealing potential contacts for cooperativity and allostery. Based on molecular dynamics (MD) simulations, small-angle X-ray scattering (SAXS) measurements, and live-cell imaging, we propose a model where the domain-swapped conformation positions the charges of the linker region to drive an interaction with the regulatory segment that modulates the degree of autoinhibition. Our findings provide a framework for understanding allosteric regulation of CaMKII by the linker region in Ca²⁺-sensitive cells.

Fig. 1. More than the length of the variable linker region affects Ca²⁺/CaM sensitivity.

A Overview of CaMKIIα holoenzyme organization containing the linker region that is made of exon 14, exon 15, and exon 18. B Subunit architecture and sequences of exon 14 and exon 18 in the variable linker region of CaMKIIα, CaMKIIβ, and CaMKIIδ. Kinase assays of CaMKIIα (C), CaMKIIβ (D), and CaMKIIδ (E) comparing the sensitivity to Ca²⁺/CaM between CaMKII with exon 14 and with exon 18 as the linker region. Each kinase assay comparison was done on the same day. Each data point represents the mean ± SD, n = 3 technical replicates. Source data are provided as a Source Data Fig. 1.

Fig. 2. Positions of charged residues on the linker dictate Ca²⁺/CaM sensitivity.

A Amino acid sequences of exon 14, exon 18, and the charge-reversal mutants. Basic and acidic residues that are mutated are colored blue and red, respectively. B, C CaMKII activity against syntide-2 is tested using linker mutants varying net charge. D CaMKII activity against syntide-2 is tested using WT exon 18 and the reverse sequence of exon 18. E Cartoon representing combinations of exon 14 and exon 18 used as the CaMKIIα variable linker region. Net charges and EC₅₀ values from activity assays shown in (F) are listed. Each kinase assay comparison was done on the same day. Each data point of each graph represents the mean ± SD, n = 3 technical replicates. Source data are provided as a Source Data Fig. 2.

Fig. 3. SEC-SAXS reveals a significant shape difference between CaMKIIα containing exon 14 or exon 18.

A, B Size-exclusion chromatographs of CaMKIIα exon 14 and exon 18 are shown. The protein was monitored with absorbance at 280 nm as shown in black trace. Evolving factor analysis (EFA) indicates the main eluent component under the peak, which is shown for exon 14 (blue) and exon 18 (red). C, D SAXS profiles of CaMKIIα exon 14 and exon 18. Insets are the Guinier analysis to interpolate the R_g value ± SD of the fit for each construct. E Pair distance distribution of SAXS profiles of CaMKIIα exon 14 and CaMKIIα exon 18 were calculated in GNOM program in RAW v.2.1.4. Source data are provided as a Source Data Fig. 3.

Fig. 4. A FRET-based assay in mouse oocytes reports a change in Ca²⁺ responsiveness comparing CaMKIIα exon 18 to CaMKIIα exon 14.

Representations of Camui exon 14 (A) and Camui exon 18 (B). For clarity, only one pair of CFP and YFP is shown. C Baseline FRET signal within the first minute of oocytes expressing either Camui exon 14 or Camui exon 18. The number within each bar is the total number of oocytes per condition. Data is shown as mean ± SD. A two-tailed, unpaired t test with Welch’s correction was performed. YFP/CFP ratios (reporting on conformational changes) and Rhod-2 traces (reporting on cytosolic Ca²⁺ level) in mouse eggs expressing Camui exon 14 (D, n = 10/2; F, n = 13/2; H, n = 11/2) and expressing Camui exon 18 (E, n = 10/2; G, n = 14/2; I, n = 13/2). n is total number of oocytes/the number of biological replicates. Source data are provided as a Source Data Fig. 4.

Fig. 5. The CaMKIIα-0 holoenzyme is comprised of domain-swapped dimers.

A Surface representation of CaMKIIδ holoenzyme crystal structure (PDB code: 8USO) in two perspectives showing the interaction between the kinase domain of one subunit and the hub domain of another subunit. B The connector segments (defined as residues 305–314) in the CaM footprints of two domain-swapping subunits are shown as sticks. The kinase domain of subunit B has been omitted for clarity.

Fig. 6. Two mutations are necessary to form predominant dimer or monomer populations.

A The dodecameric CaMKIIδ hub from the crystal structure presented here is shown with the kinase domains omitted for clarity. Residues highlighted on the right (F368, L405, Q407) are positioned in the lateral hub interface and on the bottom (C399 and H439) are positioned in the equatorial hub interface. B Mass photometry analyses at different concentrations of CaMKIIδ holoenzymes with mutations at either or both hub interfaces. Cartoon representations on the right show the positions of the mutations (indicated as X). The kinase domains are translucent for clarity. Data are represented as mean molecular weight ± SD, n = 3 technical replicates. Source data are provided in Supplementary Fig. 4.

Fig. 7. Specific electrostatic interactions between the linker and the calmodulin footprint drive the Ca²⁺/CaM sensitivity.

A Snapshots from the MD simulation at time 0 and 33 ns containing the domain-swapped dimer (shown in inset) and exon 18 linker region. Electrostatic interactions are shown using dashed lines in the right panel. B Distributions of distance between K301 of subunit B and position 316 of subunit A from simulations of constructs with different linkers as labeled. C Same as B except comparing K301 of subunit A and position 316 of subunit B. See also Supplementary Figs. 6–11. (D) Sequences of exon 18 mutants are shown to test the position of neutral or charged residues. Net charge and EC₅₀ values (see E) are shown. F Sequences of exon 14 mutants are shown to test the position of neutral or charged residues. Net charge and EC₅₀ values (see G) are shown. The net charge per residue (NCPR) graphs were plotted using CIDER server (http://157.245.85.131:8000/CIDER/). Data are shown as mean ± SD, n = 3 technical replicates. Source data are provided as a Source Data Fig. 7.