Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

Domain: chemistry
Published: 2025-12-24
Editorial outcome: major_revision

Abstract
CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of α and β isoforms in the brain. Previous biochemical studies reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKII α/β heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employ high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKII α/β at a 3:1 ratio. Our findings reveal that the α and β subunits are intermixed within the 12-meric ring assembly, with a probability exceeding 83% that β subunits are positioned adjacently. Furthermore, in the activated state, CaMKII α/β heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKII β subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKII β subunits within the CaMKII α/β heterododecamer.

Introduction
CaMKII is a holoenzyme that is abundant in neurons of the brain, particularly in postsynaptic regions, and is essential for the induction of long-term potentiation (LTP) and long-term depression (LTD), both of which serve as cellular models of learning and memory1–7. Knockout mice lacking the α isoform of CaMKII (CaMKIIα) exhibit deficits in place memory8, indicating a direct relationship with memory processes9. The β isoform (CaMKIIβ) is also abundantly expressed in neurons, and together with CaMKIIα, it plays critical roles in regulating brain function7,10,11. Recent studies have associated perturbations in CaMKIIβ expression with various neuropsychiatric and neurodevelopmental disorders9,10,12.

CaMKIIα and CaMKIIβ each consist of a kinase domain, an autoinhibitory-regulatory segment, a linker region, and a hub domain, and are activated by Ca2+/CaM binding (Fig. 1a and Supplementary Fig. 1)13–17. This binding releases the autoinhibitory-regulatory segment from the kinase domain, thereby exposing the catalytic site and the primary phosphorylation site (Thr286 in CaMKIIα or Thr287 in CaMKIIβ), which facilitates trans-autophosphorylation within the oligomer (Supplementary Fig. 1). Upon subsequent Ca2+/CaM dissociation, a secondary phosphorylation site (Thr305/306 in CaMKIIα or Thr306/307 in CaMKIIβ) becomes accessible, allowing further autophosphorylation. These phosphorylation modifications are reverted to the basal state through dephosphorylation by phosphatases (Supplementary Fig. 1). Although CaMKIIα and CaMKIIβ share this activation mechanism, their key distinction lies in linker length (CaMKIIα: 33 amino acids (a.a.), CaMKIIβ: 96 a.a., Fig. 1 and Supplementary Fig. 2). The linker in CaMKIIβ is also longer than in the γ and δ isoforms, and interspecies differences in CaMKII are largely confined to this region18–20. Structurally, electron microscopy has revealed that the homooligomers of both recombinant CaMKIIα21 and CaMKIIβ22 predominantly form a 12-meric ring assembly, in which the central hub organizes subunits through their hub domains, with kinase domains radiating outward (Fig. 1b).

Biochemical studies have shown that the relative abundance of CaMKII isoforms varies across brain regions. In the adult forebrain, the α:β ratio is approximately 3:1, whereas in the cerebellum it is reversed to 1:4 (refs. 23–27). Developmental changes have also been reported, with a near 1:1 ratio in the forebrain during the first 10 days after birth28,29. However, whether these ratios arise from heterooligomers in a CaMKII 12-mer or reflect different ratios of homooligomers of CaMKII remains a topic of debate.

How does the composition of the 12-meric ring assembly of CaMKII, as a heterooligomer or homooligomer, influence its biological function in neurons? Previous studies have indicated that only CaMKIIβ binds to F-actin via its linker region30, a property critical for neuronal localization31–33. If the CaMKIIα/β heterooligomer contains a smaller number of CaMKIIβ subunits, as observed in the forebrain, the binding of the CaMKIIα/β heterooligomer to F-actin is expected to be relatively weak, since only three CaMKIIβ subunits in the 12-mer can bind. This suggests that additional mechanisms are required for their effective localization. In contrast, in the cerebellum, where CaMKIIβ is more abundant, assemblies containing 9–10 β subunits are expected to bind strongly to F-actin. If CaMKII forms only homooligomers, CaMKIIα and CaMKIIβ would localize independently, with F-actin binding restricted to CaMKIIβ. In the context of structural plasticity of the spine, the stimulation of LTP and LTD is thought to induce the binding and dissociation of CaMKII from F-actin, leading to substantial changes in spine volume34,35. Therefore, direct evidence of the subunit composition of the 12-meric ring assembly of CaMKII could clarify its binding mode with F-actin and advance our understanding of the molecular mechanism underlying synaptic plasticity. However, thus far, biochemical experiments have only indirectly verified the stoichiometry of CaMKII variants within CaMKII oligomers through electrophoresis24, leaving the question of hetero- versus homo-oligomer composition open to speculation.

High-speed atomic force microscopy (HS-AFM) is a unique technique that allows direct visualization of the nanometer-scale dynamics of functional proteins36,37, protein‒DNA complexes38, and individual subunits within oligomers39 under liquid conditions. Previously, we applied HS-AFM to CaMKIIα homooligomers and revealed activation state-dependent structural dynamics of the kinase domain at the sub-molecular level39.

In this work, we employ HS-AFM to directly visualize the dynamics of individual subunits within a CaMKIIα/β heterooligomer with a 3:1 α:β ratio, as well as a CaMKIIβ homooligomer within a 12-meric ring assembly. Our findings provide insights into the dynamics of individual subunits in CaMKII 12-meric ring assembly and suggest biological roles on the basis of these dynamics.

Results
Kinase domains of CaMKIIβ exhibit greater mobility
We previously characterized the CaMKIIα subunit in homooligomers by measuring the distance (Dh-k) from the central hub assembly to each kinase domain and the mobility (Rg in 45 seconds) of each kinase domain39. Since CaMKIIβ possesses a longer linker than CaMKIIα does, it is possible to distinguish the CaMKIIα and CaMKIIβ subunits in the heterooligomer by comparing Dh-k and Rg values. Therefore, we initially applied HS-AFM to the CaMKIIβ homooligomer to characterize the dynamics of the β subunits. For single-molecule observation, we overexpressed CaMKIIβ in HEK293 cells and purified it using two different tags (i.e., his and strep tag), following the strategy used previously for CaMKIIα (Supplementary Fig. 3a)39. The purified CaMKIIβ was then immobilized on a positively charged pillar–arene substrate (C2P[5]A+)39,40 to allow visualization of the dynamics of the kinase domains (see the “Methods” and Supplementary Fig. 4). By selecting the AFM substrate and buffer conditions, we are able to observe CaMKII molecules in which the hub domain is stably attached to the AFM substrate, while the kinase domains exhibit dynamic movements involving weak interactions with the AFM substrate. The resulting HS-AFM images of the CaMKIIβ homooligomers revealed a gear-like structure with six protrusions at the center surrounded by scattered spherical domains (Fig. 1c and Supplementary Movie 1). Comparison with previous electron micrographs of CaMKIIβ22 indicates that the gear-like structure corresponds to the hub assembly, while the surrounding domains represent the kinase domains.

To quantify the mobility of the kinase domains of the β subunits, we calculated Dh-k and Rg on the basis of the coordinates of the center of the hub assembly and the maximum height of each kinase domain (see the “Methods”). We found that the kinase domain of CaMKIIβ in the basal state presented an average Dh-k of 15.2 ± 2.5 nm and an Rg of 3.3 ± 1.2 nm (Fig. 1d, e). The Dh-k values were consistent with those of previously reported electron micrographs of CaMKIIβ22. Furthermore, the Dh-k and Rg values were significantly greater than observed in our HS-AFM results for CaMKIIα (Fig. 1d, e). These findings suggest that the length of the linker (α: 33 a.a., β: 96 a.a.) plays a major role in determining the positioning and mobility of the kinase domains.

In addition, HS-AFM observations in the presence of bosutinib, an ATP-competitive inhibitor of CaMKII, revealed significant reductions in both Dh-k and Rg values (Fig. 1d, e, Supplementary Fig. 4h and Supplementary Movie 2). This finding suggests that, beyond linker length, interactions between the regulatory segment and the kinase domain are also important for kinase domain mobility (a stronger interaction of the regulatory segment correlates with decreased mobility of the kinase domains). In the presence of ADP or ATP, Rg did not significantly differ from that in the control experiment. These findings indicate that bosutinib, which has a stronger binding affinity than nucleotides do, enhances binding to the active site of the regulatory segment, resulting in reduced kinase domain mobility (Fig. 1d, e). Thus, while bosutinib exerts a similar inhibitory effect on both CaMKIIα and CaMKIIβ, their distinct Dh-k and Rg signatures allow the two subunits to be discriminated within CaMKIIα/β heterooligomers.

CaMKII 12-meric ring has α and β subunits with β adjacent
In forebrain neurons, biochemical studies have reported an α and β ratio of 3:1 (refs. 23, 24). To mimic this composition, we overexpressed CaMKII variants in HEK293 cells and purified them with a ratio of ~3.5:1 (see the “Methods”; Supplementary Fig. 3b, c) for HS-AFM. HS-AFM images revealed a 12-meric ring assembly resembling the structures observed for the CaMKIIα and CaMKIIβ homooligomers (Fig. 2 and Supplementary Movie 3).

We analyzed the Dh-k and Rg values for each kinase domain in the CaMKIIα/β heterooligomers to identify which kinase domains in the 12-meric ring assembly corresponded to the α and β subunits. The identification criteria are shown on the right side of Fig. 2c (see the “Methods” for details). Notably, distinguishing CaMKIIβ subunits solely on the basis of Dh-k values was challenging. The mobility of the kinase domains, as indicated by Rg, was critical for accurately identifying the CaMKIIβ subunit. The results indicated that CaMKIIα and CaMKIIβ form heterooligomers intermixed at a ratio of approximately 3:1 within the 12-meric ring assembly (Fig. 2c). This ratio showed considerable variation (Fig. 2c and Supplementary Fig. 5a).

Interestingly, when two or more CaMKIIβ subunits were present in the 12-meric ring assembly, they were more likely to be adjacent rather than randomly positioned, with probabilities of approximately 45% (18.2% for random positioning) and 100% (49.1% for random positioning) for two and three CaMKIIβ subunits, respectively, resulting in an overall probability of 83.8% (n=37 oligomers) (Supplementary Fig. 5b, c).

To further confirm the ratio and positioning of the CaMKIIβ subunit in the 12-meric ring assembly, we prepared GFP-CaMKIIβ by fusing GFP to the N-terminus of the CaMKIIβ and purified a CaMKIIα:GFP-CaMKIIβ heterooligomer with a ratio of ~4.5:1 (see the “Methods”; Supplementary Fig. 3). In this case, the CaMKIIβ subunit was labeled with GFP (small sphere), allowing for visual recognition by HS-AFM. Under our AFM experimental conditions, GFP did not strongly adsorb to the AFM substrate and appeared as a highly flexible protrusion (green arrow in Fig. 2b and Supplementary Movie 4).

As expected, the CaMKII heterooligomer included the GFP-fused kinase domain (CaMKIIβ subunit), facilitating the determination of the α:β ratio and localization. Similar to the conditions without GFP, the CaMKIIα and GFP-CaMKIIβ subunits were intermixed in the 12-meric ring assembly at a ratio of approximately 3:1 (Fig. 2d), and the probability of adjacent GFP-CaMKIIβ subunits was 78% (n=40 oligomers) (Supplementary Fig. 5b, c).

Activated CaMKIIβ kinase domains form a complex
Next, we examined the effect of the adjacent CaMKIIβ subunit in the 12-meric ring assembly on the active state of CaMKII. Initially, we premixed the CaMKIIβ homooligomer with Ca2+/CaM in tubes and performed HS-AFM to characterize the binding state of Ca2+/CaM in the CaMKIIβ subunit. An additional globular structure was observed near the kinase domain (Fig. 3a and Supplementary Movie 5). Given that the Ca2+/CaM binding site of CaMKIIβ lies close to the kinase domain (Fig. 1a) and these globular structures dissociated during HS-AFM scanning, we concluded that they correspond to Ca2+/CaM bound to the CaMKIIβ subunits.

The Dh-k value of the Ca2+/CaM-bound kinase domain was 17.9 ± 2.7 nm, which was 2.7 nm greater than that of the control, while the Rg value was 2.1 ± 1.2 nm, which was significantly lower than that of the control (Fig. 3d, e). Similarly, in the CaMKIIα/β heterooligomer, the Ca2+/CaM-bound kinase domain exhibited an extended structure (Supplementary Fig. 6a and Supplementary Movie 6). Our previous HS-AFM results of CaMKIIα homooligomers also revealed an extended structure of the kinase domain owing to Ca2+/CaM binding, with a length of approximately 3 nm (ref. 39). Thus, although CaMKIIβ possesses a linker nearly three times longer than that of CaMKIIα, both isoforms share a common activation mechanism in which Ca2+/CaM binding releases the regulatory segment from the kinase domain.

Interestingly, we found that the binding of Ca2+/CaM increases the number of kinase domains that move together in close proximity for extended periods (>45 s during HS-AFM observation) (blue box at 19.2 s in Fig. 3a). We refer to this phenomenon as a kinase domain complex (see the “Methods”). Such complexes were also observed in the basal state (Supplementary Fig. 7a); however, the binding of Ca2+/CaM enables this complex to persist even longer (Fig. 3f). Although kinase domain complexes can form in CaMKIIα subunits, they are more prominently observed in CaMKIIβ, where they occur at a higher frequency (Supplementary Fig. 7b, c).

Previous studies using single-particle electron microscopy on CaMKIIα21 and CaMKIIβ22 homooligomers, crystallographic analysis of the Caenorhabditis elegans CaMKII (CeCaMKII) kinase domain17, and various biophysical approaches41,42 have reported the formation of kinase domain dimer pairs both in vitro and in vivo43,44. Our HS-AFM observations extend these findings by directly visualizing the dynamic behavior of kinase domains, providing clear evidence of kinase domain complex formation. Importantly, these complexes exhibit highly dynamic assembly and disassembly patterns, transitioning to a more stable state with Ca2+/CaM binding, leading to the long-lasting complex state (Fig. 3).

CaMKIIβ kinase domain is more stable in pT287/pT307
Upon Ca2+/CaM binding, the first autophosphorylation occurs at Thr286 in CaMKIIα via ATP hydrolysis (Supplementary Fig. 1). We first tested the phosphorylation states of both CaMKIIα and CaMKIIβ isoforms by biochemical experiments. The results showed that CaMKIIα is phosphorylated only at Thr286, as previously reported39, while CaMKIIβ is phosphorylated at two sites (Thr287 and Thr307) (Supplementary Fig. 8). To visualize the structural dynamics of these phosphorylation states, we performed HS-AFM following the premixing of Ca2+/CaM and ATP in a tube. The phosphorylation state of CaMKIIβ under these experimental conditions was confirmed by biochemical experiments (Supplementary Fig. 8).

HS-AFM imaging of the CaMKIIβ homooligomer in the presence of Ca2+/CaM and ATP showed Ca2+/CaM bound to the proximal site of the kinase domain, as observed in the absence of ATP. The Dh-k value was 17.6 ± 2.4 nm, representing a 2.4 nm extension compared with that of the control, whereas the Rg was 2.6 ± 1.2 nm, significantly lower than that observed in the control (Fig. 3b, d, e, and Supplementary Movie 7). Our previous results for CaMKIIα homooligomers demonstrated that Ca2+/CaM+pT286 adopts an extended structure of approximately 3 nm. However, while the Rg value was comparable to that in the basal state for CaMKIIα, it remained low in CaMKIIβ.

Notably, our biochemical analyses demonstrated that phosphorylation at Thr306 (β: Thr307) occurs more efficiently in CaMKIIβ, as shown by the stronger signal in Lane 5 compared with Lane 2 in Supplementary Fig. 8. These findings are consistent with those of previous studies on an alternatively spliced variant of CaMKIIβ containing a longer linker (approximately 200 a.a.)20, which also displayed a stronger phosphorylation signal at pT307. Detailed structural comparisons of the Ca2+/CaM-bound states of CaMKIIα and CaMKIIβ, observed by HS-AFM, revealed that CaMKIIβ adopts a conformation with an enlarged gap (approximately 6 nm) between the hub and Ca2+/CaM-bound kinase domains (Supplementary Fig. 6e). This increased separation, which is absent in CaMKIIα, likely provides the additional conformational space necessary for either cis or trans kinase interactions to reach the Thr307 phosphorylation site. Such a spatial gap may therefore underlie the preferential phosphorylation of Thr307 in CaMKIIβ.

Furthermore, the number of kinase domains that remained in the complex state for a long time significantly increased in the presence of Ca2+/CaM and ATP (blue boxes at 3.9 s in Fig. 3b, f). This observation suggests that Ca2+/CaM+pT287/pT307 of CaMKIIβ forms a more stable kinase domain complex. This complex structure is likely connected to the central hub assembly through multiple β subunits, resulting in reduced mobility (lower Rg values).

We also observed the formation of kinase domain complexes between adjacent CaMKIIβ subunits in the 12-meric ring assembly of the CaMKIIα/β heterooligomers (Supplementary Fig. 6b). Most of these complexes were stable and persisted for an extended period (>45 s) (69.8%, n=43 complexes) (Supplementary Movie 8). The occurrence of kinase domain complexes between CaMKIIβ subunits in the heterooligomers suggests that the kinase domain of the CaMKIIβ subunit plays a role in stabilizing the active conformation over time.

Fully phosphorylated CaMKIIβ are both mobile and form oligomer
We next examined the structural dynamics of the kinase domains in further phosphorylated states (CaMKIIβ: pT287/pT306/pT307, Supplementary Fig. 1). CaMKIIβ homooligomers were incubated with Ca2+/CaM and ATP at 30 °C for 5 min, followed by the addition of EGTA to dissociate Ca2+/CaM, after which HS-AFM was performed. The phosphorylation state of CaMKIIβ under these experimental conditions was confirmed by biochemical experiments (Supplementary Fig. 8).

The Dh-k value of the fully phosphorylated state of the CaMKIIβ homooligomer was 17.5 ± 3.5 nm, corresponding to a significant extension of 2.3 nm relative to the control. The Rg value was 3.7 ± 2.0 nm, indicating a wide distribution (Fig. 3c–e). HS-AFM videos demonstrated that some CaMKIIβ subunits moved far from the central hub assembly and exhibited extensive movement, whereas other subunits showed low mobility, as multiple kinase domains assembled together (Fig. 3c, Supplementary Fig. 6d, and Supplementary Movie 9). Similar conformational changes in kinase domains were observed in CaMKIIα homooligomers, characterized by increased mobility and kinase domain oligomerization (KD-oligomerization)39. Thus, in the fully phosphorylated state, the conformational changes in individual CaMKIIα and CaMKIIβ subunits are similar. It is likely that phosphorylation at multiple sites disrupts the structure of the dissociated regulatory segment, resulting in increased flexibility. However, when multiple kinase domains are in close proximity due to high flexibility, some kinase domains may undergo conformational changes that lead to the entanglement of disordered regions, resulting in KD-oligomerization and decreased mobility. Stable kinase domain complexes were also observed, although at a lower frequency than in the Ca2+/CaM-bound pT287/pT307 state (blue boxes at 15.9 s in Fig. 3c, f).

HS-AFM of the fully phosphorylated state in the CaMKIIα/β heterooligomer revealed similar structural changes in both the α and β subunits (Supplementary Fig. 6c and Supplementary Movie 10). Based on these results, the differences observed between the α and β subunits in the activated state can be attributed to the formation of a stable kinase domain complex among the β subunits, facilitated by Ca2+/CaM binding. Interestingly, in the CaMKIIα/β heterooligomers, stable kinase domain complexes between β subunits frequently formed because the β subunits were initially positioned adjacent to each other in the 12-meric ring assembly.

Adjacent β reduce activity but not dephospho resistance
How does the kinase domain complex affect CaMKII function? To address this question, we performed biochemical experiments to assess the kinase activity of both CaMKII isoforms and their resistance to phosphatase-mediated dephosphorylation. Kinase activity was measured on the basis of the phosphorylation of the substrate Syntide-2 (see the “Methods”). Interestingly, in Ca2+/CaM-bound pT286 (pT287 in CaMKIIβ), kinase activity was significantly lower for CaMKIIβ homooligomers, even though the phosphorylation level of Thr286 (Thr287 in CaMKIIβ) was comparable across CaMKII isoforms and heterooligomers (Fig. 4a–c).

In addition, the resistance to dephosphorylation by a phosphatase (PP2A) was similar for CaMKIIα and CaMKIIβ homooligomers in the Ca2+/CaM-bound pT286 (pT287 in CaMKIIβ) state (Fig. 4d–f). Together with the HS-AFM findings regarding kinase domain complex formation in CaMKIIβ subunits in the activated state, these biochemical analyses indicate that the kinase domain complex likely adopts a structure that reduces kinase activity without affecting dephosphorylation resistance at Thr287.

We next sought to elucidate the possible dimeric configuration of the CaMKIIβ kinase domain in the activated state using AlphaFold3 (AF3)45. To date, the activated state of the CaMKII kinase domain has been crystallographically characterized only for the human CaMKIIδ monomer (CaMKIIδ/Ca2+/CaM complex, truncated CaMKIIδ residues 1–333; PDB: 2WEL)41. First, we used AF3 to predict the structure of the activated state of the CaMKIIβ kinase domain (residues 1–287, excluding the autoinhibitory segment). The predicted structure closely matches the previously reported structure of the CaMKIIδ/Ca2+/CaM kinase domain41, supporting that AF3 is capable of accurately modeling the activated state of CaMKII (Supplementary Fig. 7d, e). Furthermore, using this model, we predicted the dimeric structure of the activated CaMKIIβ kinase domain. The predicted dimeric structure revealed that one part of the kinase domain covers the active site of the other kinase domain (Fig. 4i). However, the pT287 site was found to be free, adopting a conformation accessible to phosphatases.

In addition, we performed protein‒protein docking on the CaMKIIδ/Ca2+/CaM complex using ClusPro46–50. The docking results suggest a stable dimer in which the catalytic sites face inward, effectively masking the essential residue for the catalytic activity (K43), while interstitial gaps allow access to the target binding site (T-site) surface (Supplementary Fig. 7f). This predicted dimer configuration aligns well with our AF3-predicted activated structure of the CaMKIIβ dimer. Therefore, the kinase domain complex predicted by AF3 can be interpreted as reducing kinase activity, as the active sites of the kinase domains mask each other, whereas the phosphorylation site (Thr287) remains exposed.

Activated kinase complexes decay slower after Ca2+/CaM dissociation
An additional potential role of the Ca2+/CaM binding-dependent kinase domain complex may involve structural stabilization of the extended structure of the kinase domain. This structure exposes the active site, as well as the substrate binding site (S-site) and T-site, enabling interactions with other proteins (Supplementary Fig. 1).

To examine the structural role of the kinase domain complex, we compared the decay time of the extended kinase domain conformation back to the basal state between CaMKIIα and CaMKIIβ. Here, we used CaMKIIαT286A and CaMKIIβT287A mutants to demonstrate that the decay of the extended structure is independent of phosphorylation. Specifically, CaMKIIαT286A or CaMKIIβT287A homooligomer was mixed with Ca2+/CaM in a tube and incubated at 30 °C for 5 min to induce the Ca2+/CaM binding state. HS-AFM was subsequently performed to observe spontaneous Ca2+/CaM dissociation. Decay time was defined as the interval between the frame in which Ca2+/CaM dissociated and the frame in which the kinase domain returned to its basal state (see the Methods; Fig. 5, Supplementary Fig. 9 and Supplementary Movie 11).

The results showed that the proportion of kinase domains that did not return to the basal state during the HS-AFM observation time (>30 s) was higher in CaMKIIβT287A (α: 22.2% [n=41], β: 66.7% [n=45] in Fig. 5g), and most of these domains had formed kinase domain complexes. This finding indicates that the kinase domain complex helps to maintain the extended structure (activated state) of the kinase domain for prolonged periods.

Methods
DNA plasmids
The rat CaMKIIα, CaMKIIαT286A and Calmodulin genes were prepared previously39. Regarding PP2A-related genes56, human PPP2CA, human PTPA (PPP2R4) and Mini-A (PPP2R1A) were also prepared previously39. Plasmids containing the CaMKIIβ gene (UniProt: P28652) were gifts from Tobias Meyer (Addgene plasmid #21227). sfGFP-CaMKIIβ and sfGFP-Mini-A were constructed by inserting sfGFP (ref. 57) and the corresponding components into the modified pEGFP-C1 vector by replacing EGFP (Clontech). For all the constructs except the Calmodulin construct, 6× His/Strep tags (MDYKDDDDHHHHHH KWSHPQFEKGTGGQQMGRDLYDDDDKDLYKSGLRSRA) were fused to the N-termini of the respective genes and inserted into the modified pEGFP-C1 vector by replacing EGFP (Clontech). CaMKII mutants with point mutations were constructed using a QuikChange site-directed mutagenesis kit (Agilent Technologies, USA). For the 6× His-tagged CaM and sfGFP-fused Syntide-2 peptide (amino acid sequence PLARTLSVAGLPGKK) constructs, the respective genes were inserted into the pRSET bacterial expression vector (Invitrogen, USA). For these constructs, a 6× His tag (MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRSEFG) was fused to the N-termini of the respective genes.

Purification of CaM and sfGFP-Syntide-2 from bacteria
His-tagged calmodulin and sfGFP-fused Syntide-2 were expressed in Escherichia coli (DH5α)39. Briefly, the cell pellet was dissolved in PBS with 1% Triton X-100 and 5 mM imidazole, sonicated, and then centrifuged. The supernatant was loaded onto a Ni2+-nitrilotriacetate (NTA) column (HiTrap, GE Healthcare, USA). The NTA column with the bound protein was washed with 5 mM and 50 mM imidazole containing 20 mM Tris/150 mM KCl, pH 7.4 buffer, and eluted with 20 mM Tris-HCl/500 mM imidazole buffer. The concentration of the purified protein was measured using the Bradford assay (Bio-Rad, USA) with BSA as a standard. The resulting protein concentration was typically ~500 µM in a total volume of 1000 μl. The purified proteins were stored in 20 mM Tris-HCl/150 mM KCl with 5 mM DTT at 4 °C or −30 °C.

Purification of CaMKII and PP2A from HEK293 cells
Briefly, 6× His/Strep-tagged CaMKII proteins were expressed in HEK293 cells (ATCC, CRL-11268)39. The cells were transfected with a plasmid encoding the CaMKIIα/β genes using Lipofectamine 2000 following the manufacturer’s protocol (Thermo Fisher Scientific, USA). For the CaMKIIα/CaMKIIβ and CaMKIIα/sfGFP-CaMKIIβ complexes, two plasmids were cotransfected at a ratio of ~3 (CaMKIIα) to 1 (CaMKIIβ or sfGFP-CaMKIIβ). One day after transfection, the cells were lysed with ice-cold lysis buffer (1% Triton X-100, 5% glycerol, 50 mM Tris, 150 mM KCl, and 4 mM EDTA, pH adjusted to 7.6) and then centrifuged at 20,000 × g for 10 min. The supernatant was loaded onto Strep-Tactin Sepharose (Nacalai, Japan), washed with 20 mM Tris-HCl/150 mM KCl, and subsequently eluted with 2.5 mM desthiobiotin/20 mM Tris-HCl/150 mM KCl, pH 7.4. The pooled proteins were further purified with an NTA column, and the protein concentration was determined using the Bradford assay. The resulting protein concentrations typically ranged from 1~10 µM in a total volume of 500 μl. The purified proteins were stored in 20 mM Tris-HCl/150 mM KCl with 50% glycerol and 5 mM DTT, pH 7.4, at −30 °C.

For PP2A, three plasmids encoding PPP2CA/PTPA/sfGFP-Mini-A genes were cotransfected at a ratio of 1:1:1. One day after transfection, the cells were lysed with ice-cold lysis buffer (1% Triton X-100, 5% glycerol, 50 mM Tris, 150 mM KCl, 0.5 mg/ml BSA, 1 mM ATP, pH adjusted to 7.6) and then centrifuged at 20,000 × g for 10 min. The supernatant was loaded onto an NTA column, and the bound protein was washed with buffer (20 mM Tris/150 mM KCl/5% glycerol/0.5 mg/ml BSA/1 mM ATP, pH 7.4) containing 50 mM imidazole and eluted with buffer containing 500 mM imidazole. The PP2A activity was determined by previously reported methods39. The resulting PP2A activity was typically ~0.2 unit/µl in a total volume of 500 μl. The purified proteins were stored in 20 mM Tris/150 mM KCl/50% glycerol/5 mM DTT/0.5 mg/ml BSA/1 mM ATP, pH 7.4, at −30 °C.

Kinase activity assay
A kinase assay was performed at 30 °C for 5 min of incubation with 50 nM purified CaMKII protein, 800 nM CaM, 1 mM CaCl2, and 1 mM ATP in reaction buffer (50 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM MgCl2). To release CaM from CaMKII, 2 mM EGTA was added, and the mixture was incubated for 5 min at 30 °C and for an additional 10 min at 25 °C to match the conditions for AFM observation. For substrate phosphorylation, sfGFP-Syntide-2 was added at 1 μM followed by incubation for 7 min at 25 °C. For CaMKII dephosphorylation by PP2A, phosphorylated CaMKII proteins were incubated with the indicated units of PP2A at 25 °C. The reactions were stopped by the addition of SDS sample buffer.

Western blotting was performed using the following antibodies: anti-phospho-CaMKII (Thr286) (D21E4; Cell Signaling Technology, USA), anti-phospho-CaMKII (Thr305) (abx012403; Abbexa), anti-phospho-CaMKII (Thr306) (p1005-306; PhosphoSolutions), anti-phospho-PKA substrate (RRXS*/T*) (#9624; Cell Signaling Technology), anti-His-tag (27E8; Cell Signaling Technology), anti-pan-CaMKII (D11A10; Cell Signaling Technology), anti-GFP (M048-3; MBL), anti-CaMKIIβ (ab34703; Abcam), anti-CaMKIIα (13730-1-AP; Proteintech), and HRP-conjugated anti-rabbit/mouse secondary antibodies (Jackson ImmunoResearch Laboratories). All primary/secondary antibodies were used at a 1:4000 dilution, except for anti–His-tag (27E8), which was used at a 1:5000 dilution.

HS-AFM equipment
HS-AFM experiments were conducted with a custom-designed instrument in tapping mode38,39. The deflection of the cantilever (BL-AC10DS-A2, Olympus, Japan) was measured using an optical beam deflection sensor with an infrared (IR) laser at 780 nm and a power of 0.8 mW. The IR beam was directed onto the rear of the cantilever using a 60× objective lens (CFIS Plan Fluor ELWD 60X, Nikon, Japan). The spring constant of the cantilever was approximately 100 pN/nm, whereas its resonant frequency and quality factor in liquid were approximately 400 kHz and 2, respectively. Although the cantilever originally featured a bird beak-like triangular structure as the AFM tip, an amorphous carbon tip was synthesized on the existing AFM tip via electron beam deposition (EBD) using scanning electron microscopy (FE-SEM; Verious 5UC, Thermo Fisher Scientific, USA) to increase the spatial resolution of the HS-AFM. The length of the additional EBD tip was approximately 500 nm, and the tip apex had a radius of approximately 1 nm after further plasma etching using a plasma cleaner (Tergeo, P.I.C. Scientific, USA). The cantilever’s free oscillation amplitude was set to below 1 nm, and the setpoint amplitude for tip‒sample distance regulation was maintained at 90% of this value. Imaging acquisition and tip-scanning control were performed using laboratory-developed software written in Visual Basic.NET (Microsoft). To minimize sample damage caused by tip scanning, we employed the Only Trace Imaging mode58. In this approach, only the trace images are acquired by scanning the tip across the sample surface, whereas no image is recorded during the retrace, in which the tip is slightly lifted away from the sample and scanned at the same speed as in the trace.

Substrates for HS-AFM observations
Consistent with our previous study involving HS-AFM observations of CaMKIIα39, all the HS-AFM observations were conducted on a mica surface modified with a cationic C2 pillar[5]arene (C2P[5]A+) to change the surface charge from negative to positive40,59. Electrostatic potential mapping of CaMKIIα and CaMKIIβ revealed that the surface charge of the hub domain is partially negative, whereas the surface charge of the kinase domains is partially positive (Supplementary Fig. 4a). When we tested bare mica with a negatively charged surface as the substrate, the kinase domains of the CaMKIIβ homooligomers and CaMKIIα/β heterooligomers strongly adhered to the mica, limiting their movement because of their strong affinity for the negatively charged AFM substrate through electrostatic interactions (Supplementary Fig. 4b, c). Therefore, to prevent the inhibition of kinase domain mobility on the HS-AFM substrate, C2P[5]A+ was used as the HS-AFM substrate. An aqueous solution of 70 μM C2P[5]A+ was deposited onto a freshly cleaved mica substrate (1.0 mm in diameter; Furuuchi Chemical, Japan). C2P[5]A+ was incubated at room temperature on the mica surface for 15 min, and the surface was rinsed with Milli-Q water to eliminate unadsorbed C2P[5]A+.

HS-AFM observations
The basal states of CaMKIIα/β heterooligomers and CaMKIIα and CaMKIIβ homooligomers were observed in buffer A (50 mM Tris-HCl, pH 7.4; 15 mM KCl; 10 mM MgCl2; and 10% glycerol) with 0.1 mM EGTA. For the inhibitor experiment, 50 nM CaMKIIβ homooligomers were premixed at 30 °C for 5 min with 50 μM bosutinib in buffer B (50 mM Tris-HCl, pH 7.4; 150 mM KCl; 10 mM MgCl2) supplemented with 0.1 mM EGTA. Then, HS-AFM observations were performed in buffer A with 0.1 mM EGTA and 50 μM bosutinib. Bosutinib (Cat. No. 4361) was purchased from Tocris (UK).

For Ca2+/CaM-bound CaMKIIα/β heterooligomers and CaMKIIβ homooligomers, including the WT, CaMKIIαT286A and CaMKIIβT287A mutants, 50 nM CaMKII and 800 nM CaM were premixed in buffer B with 1 mM CaCl2 and incubated at 30 °C for 5 min. Then, HS-AFM observations were performed in buffer A with 1 mM CaCl2.

For Ca2+/CaM-bound CaMKIIα/β heterooligomers and CaMKIIβ homooligomers in the presence of ATP, we premixed 50 nM CaMKII and 800 nM CaM in buffer B with 1 mM CaCl2 and 1 mM ATP and incubated the mixture at 30 °C for 5 min. Then, HS-AFM observations were performed in buffer A with 1 mM CaCl2 and 1 mM ATP.

For CaMKIIα/β heterooligomers and CaMKIIβ homooligomers in EGTA and ATP, we premixed 50 nM CaMKII and 800 nM CaM in buffer B with 1 mM CaCl2 and 1 mM ATP and incubated the mixture at 30 °C for 5 min. Afterward, to dissociate Ca2+/CaM from CaMKII, we added 2 mM EGTA and incubated the mixture at 30 °C for an additional 5 min. Then, HS-AFM observations were performed in buffer A with 2 mM EGTA and 1 mM ATP.

Regarding the optimization of buffer composition, we added 15 mM NaCl to the imaging buffer to simulate physiological conditions inside the cytoplasm. However, the presence or absence of NaCl did not influence the dynamics of the kinase domains in either CaMKIIβ homooligomers or CaMKIIα/β heterooligomers (Supplementary Fig. 4d). Furthermore, we observed kinase domain complexes among CaMKIIβ subunits in both the basal and activated states (Ca2+/CaM+ATP, Supplementary Fig. 4e), and in the presence of 150 mM KCl (Supplementary Fig. 4f, g). We note that the dynamics at 150 mM KCl looks similar to those at 15 mM KCl. Therefore, as described in our previous study39, we conclude that while salt concentration influences the interaction between CaMKII oligomers and the AFM substrate, the conditions used in this study—including the substrate and buffer conditions—were optimized to observe the kinase domain dynamics of CaMKII and allow for comprehensive analysis.

All HS-AFM experiments were performed at room temperature (24–26 °C).

HS-AFM image processing and data analysis
HS-AFM images were analyzed using Fiji (ImageJ) software (NIH, USA)60. A mean filter with a radius of 1.0 pixels was applied to each image to minimize noise. To correct drift in sequential images, the Template Matching and Slice Alignment plugin for ImageJ was utilized. The coordinates of the hub assembly center were determined using the Trainable Weka Segmentation plugin61. The MTrackJ plugin62 for ImageJ was used for manual tracking of the coordinates corresponding to the highest pixel for each kinase domain across all CaMKII protein constructs under various HS-AFM experimental conditions.

The motion of each kinase domain for the CaMKIIα/β heterooligomer (56,856 points in 4738 frames of 528 kinase domains in 44 oligomers), the CaMKIIβ homooligomer (18,000 points in 1,500 frames of 120 kinase domains in 10 oligomers), CaMKIIβ+bosutinib (24,024 points in 2002 frames of 168 kinase domains in 14 oligomers), CaMKIIβ+ADP (19,608 points in 1634 frames of 132 kinase domains in 11 oligomers), CaMKIIβ+ATP (17,531 points in 1473 frames of 119 kinase domains in 10 oligomers), and CaMKIIβ in EGTA/ATP after Ca2+/CaM/ATP stimulation (25,512 points in 2126 frames of 192 kinase domains in 16 oligomers) was analyzed. The motion of the Ca2+/CaM-binding kinase domains in CaMKIIβ with ATP (18,000 points in 1500 frames of 120 kinase domains in 10 oligomers) and without ATP (18,000 points in 1500 frames of 120 kinase domains in 10 oligomers) was analyzed.

The motion of each kinase domain of CaMKIIαT286A (7767 points of 41 kinase domains in 22 oligomers) and CaMKIIβT287A (8393 points of 45 kinase domains in 32 oligomers) was analyzed.

HS-AFM experiments were independently repeated at least three times with similar results.

Analysis of kinase domain trajectories
To quantify the trajectory of a single kinase domain, the distance from the center of the hub assembly to the kinase domain (Dh-k) was computed as follows:

Dh-k = |~Rk(n) - ~Rhub(n)|,

where ~Rk(n) is the position of the kinase domain in video frame (n) and where ~Rhub(n) is the position of the center of the hub assembly in video frame (n).

To characterize a single kinase domain, the radius of gyration, Rg, of the kinase domain trajectory was computed as the root-mean squared displacement from its average position:

Rg2 = (1/N) Σn=1N |~Rk(n) - <~Rk>|2

where <~Rk> is the average position of a single kinase domain. Rg was calculated from HS-AFM videos over a duration of 45 s, with a temporal resolution of 0.3 s per frame.

Distinguishing α and β subunits in heterooligomers
First, the distribution of Dh-k for the CaMKIIα and CaMKIIβ homooligomers indicated that kinase domains with Dh-k values greater than 14 nm could be identified as CaMKIIβ subunits. Consequently, 97.9% (n=144 kinase domains) of the kinase domains in CaMKIIα homooligomers were identified as CaMKIIα subunits, whereas 66.7% (n=120 kinase domains) of the kinase domains in the CaMKIIβ homooligomers were classified as CaMKIIβ subunits. This suggests that 33.3% of CaMKIIβ subunits would be misidentified as CaMKIIα subunits if assessed solely on the basis of Dh-k values. To account for the remaining CaMKIIβ subunits, we introduced an additional criterion: kinase domains with an Rg value greater than 2.8 nm were classified as CaMKIIβ subunits, even if their Dh-k value was less than 14 nm. This Rg value threshold of 2.8 nm was estimated on the basis of the distribution of Rg values for CaMKIIα and CaMKIIβ homooligomers. Using this criterion, 91.7% of the kinase domains in the CaMKIIα homooligomers were identified as CaMKIIα subunits, whereas 61.7% of the kinase domains in the CaMKIIβ homooligomers were identified as CaMKIIβ subunits. By combining these two criteria (first, Dh-k values over 14 nm, and second, Rg values greater than 2.8 nm, even if the Dh-k value was below 14 nm), 98.6% of the kinase domains in the CaMKIIα homooligomers were identified as CaMKIIα subunits, whereas 80.8% of the kinase domains in the CaMKIIβ homooligomers were classified as CaMKIIβ subunits. Notably, an error of approximately 19.2% (2.3 subunits out of a 12-mer) could result in the misidentification of CaMKIIβ subunits as CaMKIIα subunits, suggesting that the actual number of CaMKIIβ subunits in CaMKIIα/β heterooligomers is likely somewhat greater. We analyzed 44 CaMKIIα/β heterooligomers. Among these, two oligomers contained no CaMKIIβ subunits, whereas five oligomers contained one CaMKIIβ subunit each. Of the remaining 37 CaMKIIα/β heterooligomers, 31 oligomers exhibited adjacent CaMKIIβ subunits. This finding indicates that CaMKIIβ subunits are adjacent to one another, with a probability of 83%, in CaMKIIα/β heterooligomers within the 12-meric ring.

Analysis of kinase domain complexes
Kinase domain complexes were defined on the basis of the distance between kinase domains and their mobility. First, the distance between the maximum heights of each kinase domain (Dk-k in Fig. 3f) was measured and found to be shorter than the length of the major axis of the kinase domains. The length of the major axis of the kinase domains was calculated in regions greater than 2 nm in height from the AFM substrate. For CaMKIIβ, the values were recorded as follows: 6.0 ± 0.7 nm (120 kinase domains in 10 oligomers) in the basal state, 6.4 ± 0.9 nm (120 kinase domains in 10 oligomers) in the Ca2+/CaM binding state, 6.2 ± 0.8 nm (120 kinase domains in 10 oligomers) in the Ca2+/CaM+pT287 state, and 6.6 ± 1.0 nm (192 kinase domains in 16 oligomers) in the pT287/pT306/pT307 state, and the corresponding values for CaMKIIα were 5.3 ± 0.6 nm (120 kinase domains in 10 oligomers) in the basal state, 6.3 ± 0.9 nm (108 kinase domains in 9 oligomers) in the Ca2+/CaM binding state, 5.7 ± 0.8 nm (120 kinase domains in 10 oligomers) in the Ca2+/CaM+pT286 state, and 5.3 ± 0.6 nm (120 kinase domains in 10 oligomers) in the pT286/pT305/pT306 state. Next, to exclude kinase domain complexes consisting of two kinase domains located close to each other in each HS-AFM frame, we used the mobility of each kinase domain to define the kinase domain complex. For example, if two kinase domains were positioned adjacent to each other and the distance between them exceeded the length of the major axis of the kinase domains, yet the kinase domains moved together in the same direction, we classified these kinase domains as being in a complex state. Conversely, if the distance between two kinase domains was less than or equal to the length of the major axis of the kinase domains, but they did not move in the same direction, these kinase domains were not classified as being in a complex state.

For the dwell time analysis of the kinase domain complex, only cases in which the complex was maintained for more than 10 frames (0.3 [s]×10 [frames]=3 [s]) were counted. In addition, when the distance between the kinase domains exceeded the length of the major axis of the kinase domain for 5 frames, it indicated the release of the kinase domain complex. For CaMKIIβ, a total of 10 oligomers (1500 frames) in the basal state, 10 oligomers (1500 frames) in the Ca2+/CaM-bound state, 10 oligomers (1500 frames) in the Ca2+/CaM+pT287 state, and 9 oligomers (1350 frames) in the pT287/pT306/pT307 state were analyzed to evaluate the dwell time of the kinase domain complex, and the corresponding numbers for CaMKIIα were 10 oligomers (1460 frames) in the basal state, 9 oligomers (1320 frames) in the Ca2+/CaM-bound state, 10 oligomers (1517 frames) in the Ca2+/CaM+pT286 state, and 10 oligomers (1484 frames) in the pT286/pT305/pT306 state.

Contraction analysis of the kinase domains
We analyzed the kinase domains from which Ca2+/CaM dissociated during HS-AFM scanning. The Dh-k values of these kinase domains were measured for approximately 100 frames before and after the dissociation of Ca2+/CaM. The dissociation of Ca2+/CaM was detected manually through frame-by-frame comparisons of the HS-AFM images. We defined contraction of the kinase domains to the autoinhibitory state as a decrease in the Dh-k value of approximately 2.0 nm or more. Kinase domains that did not exhibit any decrease in Dh-k values within 30 s after Ca2+/CaM dissociation were classified as noncontracted kinase domains.

Quantification and statistical analysis
All statistical analyses were performed using Igor Pro 9 software (WaveMetrics, USA) or OriginPro 2025 (OriginLab, USA). A significance level of α = 0.05 was applied for all analyses, and P values were adjusted for multiple comparisons where relevant. Unless otherwise stated, the normality of the data distribution was tested by the Shapiro‒Wilk test. If the normality assumption failed, the Kruskal‒Wallis test and Mann‒Whitney U test were used for comparisons between two groups. For comparisons involving multiple conditions, one-way ANOVA and the Kruskal‒Wallis test were used to analyze multiple groups with a single independent variable. As a follow-up test to the Kruskal‒Wallis test, Dunn’s test was used to compare every mean with every other mean. The Bonferroni correction was used as the follow-up test for one-way ANOVA. The tests used are indicated in the figure captions. P > 0.05, not significant (NS); *P < 0.05, **P < 0.01, and ***P < 0.001 were considered to indicate statistical significance. The data are presented as the means ± SDs.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Critique\nThe major goal of this paper is to elucidate the dynamics at nanoscale of calmodulin-dependent protein kinase (CAMKII), which is a key enzyme in the central nervous system involving in synaptic plasticity, learning, and memory. The CAMKII holoenzyme assembles as 12-mer comprising of two variants,   and  , so ratio between their loading on the full assembly defines the protein function and specifically its interaction with F-actin. The authors used time-lapse high-speed AFM (HS-AFM) capable of capturing the dynamics of macromolecules and assemblies with nanometer range resolution. They did reveal and characterize the difference in dynamics of catalytic domains with the multi-unit assemblies for both variants depending on the domain types and the ligands. Technically, the paper is well done but a number of issues remain.\n1. The major concern with this paper is the translation of the results obtained with the CAMKII function. The dynamics was studied at the surface, although the function of the protein is not associated with its binding to surfaces. Importantly, the authors used a positively charged surface, so the contribution of electrostatic interaction of the protein domains with the surface can contribute to the dynamics. What if negatively charged surface is used for the AFM experiments?\n2. In the time-lapse AFM buffer with the composition far from physiological was used. Specifically, no NaCl was added. The experiments in the buffer solution corresponding to the composition relevant to physiological are needed to translate the findings to the protein function in neurons. Additionally, experiments with various salt concentration may help evaluate the role of electrostatic interaction with the surface in the domains dynamics at the surface-liquid interface (see the concern above).\n3. To characterize the role of CAMII interaction with F-actin, simulations were used. The system is quite large, so a validation of the computer simulations results obtained is needed to justify the model proposed (Fig. 6).", "reviewer_2": "This manuscript presents an elegant study on the structural dynamics of subunits and individual domains of CaMKII in a CaMKIIa/b-heterooligomer setting. The main findings at a high-resolution recapitulation of the 3:1 ratio of a/b in a heterooligomer are that (1) HS-AFM can parametrically distinguish CaMKII a from b, owing to larger linker length of b, (2) the b subunits are preferentially adjacent in ~3:1 heteromer, (3) CaMKII forms a stable kinase domain complex in presence of Ca/CaM, with adjacent b subunits forming a tighter complex, which decrease kinase activity but does not alter dephosphorylation resistance. These are important findings for the field, and the methodology used is novel in the context of these questions for CaMKII. However, I do have some concerns about the approaches taken to arrive at some conclusions and some suggestions for clarification to strengthen the work before it can be considered for publication.\n1. One major issue is that the authors have not presented their HS-AFM findings in the context of previous literature. The kinase dimer complex is presented here as a new finding, but the idea of two adjacent kinase domains forming a dimer pair has been discussed in many past papers using cryo-EM, crystallography, and other biophysical techniques, as summarized in many reviews (e.g., PMID: 31653643). I strongly suggest that the authors present their work from the perspective of these results over the last 20 years. I very much like the HS-AFM for examining the kinetics of dimer formation or the release of Ca/CaM, which provides a temporal axis to these findings. Discussing these advantages in the context of the literature will strengthen the outcomes of this work.\n2. How does the Alphafold3 kinase dimer complex model match the available kinase domain dimer structures for camkiia? The authors should take advantage of the rich structural data that is already available (especially on kinase domains) for various camkii isoforms. A related point is that the structural details are not clear in the figure. 4g.\n3. The western blots in Fig. 4 should be presented with a loading control where both total CamKII a/b loaded and associated phospho signals are shown. Currently, no such normalization is presented for these blots.\n4. There is no data to show the quality of the purified protein under various conditions. CaMKII is notoriously prone to aggregation, especially under conditions of Ca/CaM+ATP treatments. So, a size exclusion profile to show the qualities of the representative protein used under the key conditions will be reassuring.\n5. The beta linker is said to be ~96 residues. The human b linker can be as long as ~200 residues. Which splice variant is being used? Uniprot identifiers and variant identifiers will improve data reproducibility. Also, how does changing the linker length from, say, 96 to the b variant that has a 200-residue linker change the dynamics of the kinase domain complexes?\n6. Past studies have shown differences in propensity of T286 versus 305/306 phosphorylation between a and b homooligomers. Can the observed dynamics explain these data? I think it is interesting based on the linker lengths that change the probability of camkiia versus camkiib kinases to autophosphorylate in trans versus cis, which can alter the phosphorylation probabilities at the 286/287 versus 305/306 sites. Can the authors please comment on this in the light of their data, where they can see the distances between hub and kinases distinctly for both isoforms?"}, "author_response": "Response to the reviewers’ comments and remarks\nManuscript ID; NCOMMS-25-35814-T\nWe are genuinely grateful to the reviewers for dedicating their time and effort to reviewing our\npaper. We appreciate their positive feedback and constructive insights into our research. In\nthe revised manuscript, all concerns raised by the reviewers have been addressed. We believe\nthat their contributions and suggestions have enhanced the clarity and quality of the\nmanuscript. Revised parts of the manuscript and answers to each comment included in this\ndocument are in red, and responses to reviewers’ comments are in blue.\nReviewer #1 (Comments):\nThe major goal of this paper is to elucidate the dynamics at nanoscale of calmodulin-dependent\nprotein kinase (CAMKII), which is a key enzyme in the central nervous system involving in\nsynaptic plasticity, learning, and memory. The CAMKII holoenzyme assembles as 12-mer\ncomprising of two variants, α and β, so ratio between their loading on the full assembly\ndefines the protein function and specifically its interaction with F-actin. The authors used time-lapse\nhigh-speed AFM (HS-AFM) capable of capturing the dynamics of macromolecules and\nassemblies with nanometer range resolution. They did reveal and characterize the difference\nin dynamics of catalytic domains with the multi-unit assemblies for both variants depending on\nthe domain types and the ligands. Technically, the paper is well done but a number of issues\nremain.\n1. The major concern with this paper is the translation of the results obtained with the CAMKII\nfunction. The dynamics was studied at the surface, although the function of the protein is not\nassociated with its binding to surfaces. Importantly, the authors used a positively charged\nsurface, so the contribution of electrostatic interaction of the protein domains with the surface\ncan contribute to the dynamics. What if negatively charged surface is used for the AFM\nexperiments?\nReply)\nThank you for the constructive suggestions. We entirely agree with the reviewer’s comments.\nWe have carefully examined the effect of AFM substrates on CaMKII dynamics.\nFirst, in our previous study on CaMKIIα homooligomers [Tsujioka et al. Sci. Adv. (2023)],\nwe tested bare mica, which provides a negatively charged surface. Under these conditions,\nthe majority of kinase domains adhered strongly to the AFM substrate, restricting their\nmovement. The attached electrostatic potential map of the CaMKIIα 12-mer (Supplementary\nFig. 4a) clearly indicates that the kinase domains are primarily positively charged, which\nexplains their strong affinity for the negatively charged AFM substrate through electrostatic\ninteractions.\nthe same negatively charged surface (bear mica) for the CaMKIIβ homooligomers and\nCaMKIIα/β heterooligomers. The kinase domains again adhered tightly to the surface, resulting\nin uneven distribution around the central hub assembly and preventing proper observation of\ntheir dynamics (Supplementary Fig. 4b,c). Taken together, we concluded that AFM substrates\nwith weak positive charges (C2P[5]A+) closely approximate physiological dynamics and are\noptimal for observing the natural behavior of kinase domains. We have clarified the\noptimization of the AFM substrate in the Methods section and added a new panel to\nSupplementary Figure 4 in the revised manuscript.\nP.5, L.115\nThe purified CaMKIIβ was then immobilized on a positively charged pillar–arene substrate\n(C2P[5]A+)39,40 to enable visualization of the dynamics of the kinase domains (see the Methods\nand Supplementary Fig. 4).\nP.19, L.565\n, whereas the surface charge of the kinase domains is partially positive (Supplementary Fig.\n4a). When we tested bare mica with a negatively charged surface as the substrate, the kinase\ndomains of the CaMKIIβ homooligomers and CaMKIIα/β heterooligomers strongly adhered to\nthe mica, limiting their movement because of their strong affinity for the negatively charged\nAFM substrate through electrostatic interactions (Supplementary Fig. 4b, c).\n2. In the time-lapse AFM buffer with the composition far from physiological was used.\nSpecifically, no NaCl was added. The experiments in the buffer solution corresponding to the\ncomposition relevant to physiological are needed to translate the findings to the protein\nfunction in neurons. Additionally, experiments with various salt concentration may help\nevaluate the role of electrostatic interaction with the surface in the domains dynamics at the\nsurface-liquid interface (see the concern above).\nReply)\nWe thank the reviewer for this valuable comment.\nTo address this point, we incorporated 15 mM NaCl into the observation buffer and\nperformed additional HS-AFM experiments on CaMKIIβ homooligomers. The results showed\nthat the presence or absence of NaCl did not alter the dynamics of the kinase domains\n(Supplementary Fig. 4d). Complex formation among CaMKIIβ subunits was also consistently\nobserved in both the basal state and the activated state (Ca2+/CaM+ATP) (Supplementary Fig.\n4e). Furthermore, biochemical analyses confirmed that kinase activity and phosphorylation of\nCaMKII were unaffected under our experimental conditions, even without NaCl.\n(150 mM KCl), corresponding more closely to physiological ionic strength, again using\nCaMKIIβ homooligomers. Under this condition, the kinase domains exhibited free movements\naround the hub assembly, and complex formation was observed in the activated state\n(Supplementary Fig. 4f,g), similar to the results at 15 mM KCl. However, at this high salt\nconcentration, the weakened interaction between the protein and the AFM substrate prevented\nHS-AFM from capturing the detailed movements of individual kinase domains.\nThese findings are consistent with our previous study on CaMKIIα homooligomers\n[Tsujioka et al., Sci. Adv. (2023)], where we systematically tested various salt concentrations\nto establish optimal conditions. Together, the results indicate that although salt concentration\ninfluences the interaction of CaMKII with the AFM substrate, it does not affect the intrinsic\ndynamics or complex formation of CaMKIIβ subunits.\nAccordingly, we conclude that the experimental conditions used in the present study—\nincluding the AFM substrate and buffer composition—are optimal for reproducing the natural\ndynamics of CaMKII oligomers while allowing detailed observation. We have clarified the\noptimization of these HS-AFM conditions in the Methods section and added a new panel to\nSupplementary Figure 4 in the revised manuscript.\nP.20, L. 600\nRegarding the optimization of buffer composition, we added 15 mM NaCl to the imaging buffer\nto simulate physiological conditions. However, the presence or absence of NaCl did not\ninfluence the dynamics of the kinase domains in either CaMKIIβ homooligomers or CaMKIIα/β\nheterooligomers (Supplementary Fig. 4d). Furthermore, we observed kinase domain\ncomplexes among CaMKIIβ subunits in both the basal and activated states (Ca2+/CaM+ATP;\nSupplementary Fig. 4e), supporting our conclusions regardless of the presence of NaCl.\nAdditionally, we performed HS-AFM observations using 150 mM KCl buffer for CaMKIIβ\nhomooligomers. The results indicated that the kinase domains moved freely around the central\nhub assembly (Supplementary Fig. 4f), and the formation of kinase domain complexes was\nconsistently observed in the activated state, similar to observations at 15 mM KCl\n(Supplementary Fig. 4g). At high salt concentrations, due to the weak interaction between the\nAFM substrate and the protein, the motions of kinase domains captured by HS-AFM were too\nrapid to precisely analyze the detailed movements of individual kinase domain within the\nCaMKII oligomer. We note that the dynamics at 150 mM looks similar to those at 15 mM KCl.\nTherefore, we conclude that while salt concentration influences the interaction between\nCaMKII oligomers and the AFM substrate, it does not affect the intrinsic dynamics or complex\nformation of the CaMKIIβ subunits within the oligomers. As explained in our previous study39,\nthe experimental conditions used in this study, including the AFM substrate and buffer\n3\ncomposition, were optimized to faithfully replicate the natural dynamics of CaMKII kinase\ndomains and enable detailed analysis.\nSupplementary Fig. 4: Optimization of HS-AFM observations of CaMKII oligomers\na, Electrostatic potential map of CaMKIIα, which is based on a pseudoatomic model derived\nfrom EM data21.\nb–e, HS-AFM images of CaMKIIβ homooligomers and CaMKIIα/β heterooligomers adsorbed\non mica surfaces (b, c) and under different buffer conditions (d, e). Frame rate, 3.3 frames/s.\nThe trajectories of the kinase domains and the center of the hub assembly (red in the center)\nwere tracked for approximately 40 s (also in h).\nf–g, Sequential HS-AFM images of CaMKIIβ homooligomers in the basal state (f) and under\nCa2+/CaM + ATP conditions (g) with high salt concentrations in 15 mM NaCl buffer.\nh, Sequential HS-AFM images of CaMKIIβ in the presence of 50 μM bosutinib (see also\nSupplementary Movie 2).\n3. To characterize the role of CaMKII interaction with F-actin, simulations were used. The\nsystem is quite large, so a validation of the computer simulations results obtained is needed\nto justify the model proposed (Fig. 6).\nReply)\nThank you for the constructive suggestions. The simulation size used in this study involves a\ngrid resolution of 1 nm by 1 nm, resulting in 300 × 300 = 90,000 interaction points across the\nentire system. Since the CaMKIIα/β heterooligomer model with three β subunits has three\ninteraction sites, the total number of interactions between the heterooligomer and the system\nis 3 × 90,000 = 270,000. Even in the molecular dynamics simulations of four-point water\nmodels conducted over twenty years ago—such as modeling 200 water molecules within a\ncube approximately 2 nm per side—there were 318,400 interactions, calculated as 200 × 199/2\n× 16. With current computational resources, our system size is, therefore, not exceedingly\nlarge. In fact, our calculations did not require the use of supercomputers and were performed\non a commercially available desktop computer.\nFurthermore, our mathematical modeling was based on parameters derived from\nprevious experimental data, as described in the Methods section, supporting the robustness\nof our model validation. However, we recognize that the original manuscript may not have\nsufficiently clarified the validation process of our simulation model, especially in the main text.\nTherefore, we have added a detailed explanation to the revised manuscript to clarify this point.\nP.13, L.383\nWe used a model in which three CaMKIIβ subunits were assumed to be present within a 12-\nmer, with the CaMKIIβ subunits arranged either in an equally spaced configuration (subunit-\n4\ndispersion model) or adjacent to each other (subunit-adjacent model) (Fig. 6a, b). The CaMKII\nmodel was developed based on the HS-AFM observations and electron microscopy images21,\nensuring that the structural features are consistent with the experimental data. Similarly, the\nF-actin model was constructed using structural information derived from previous high-\nresolution studies52-54. Furthermore, the interaction between F-actin and CaMKII was\nparameterized to accurately reproduce the binding affinity (K ) for CaMKIIβ and F-actin, as\na\nreported previously55 (see Methods).\nReviewer #2 (Comments):\nThis manuscript presents an elegant study on the structural dynamics of subunits and\nindividual domains of CaMKII in a CaMKIIa/b-heterooligomer setting. The main findings at a\nhigh-resolution recapitulation of the 3:1 ratio of a/b in a heterooligomer are that (1) HS-AFM\ncan parametrically distinguish CaMKII a from b, owing to larger linker length of b, (2) the b\nsubunits are preferentially adjacent in ~3:1 heteromer, (3) CaMKII forms a stable kinase\ndomain complex in presence of Ca/CaM, with adjacent b subunits forming a tighter complex,\nwhich decrease kinase activity but does not alter dephosphorylation resistance. These are\nimportant findings for the field, and the methodology used is novel in the context of these\nquestions for CaMKII. However, I do have some concerns about the approaches taken to arrive\nat some conclusions and some suggestions for clarification to strengthen the work before it\ncan be considered for publication.\n1. One major issue is that the authors have not presented their HS-AFM findings in the context\nof previous literature. The kinase dimer complex is presented here as a new finding, but the\nidea of two adjacent kinase domains forming a dimer pair has been discussed in many past\npapers using cryo-EM, crystallography, and other biophysical techniques, as summarized in\nmany reviews (e.g., PMID: 31653643). I strongly suggest that the authors present their work\nfrom the perspective of these results over the last 20 years. I very much like the HS-AFM for\nexamining the kinetics of dimer formation or the release of Ca/CaM, which provides a temporal\naxis to these findings. Discussing these advantages in the context of the literature will\nstrengthen the outcomes of this work.\nReply)\nThank you for the constructive suggestions. The formation of kinase domain dimers has been\nwidely reported in previous studies. However, this study clearly demonstrates that the kinase\ndomain complex, including dimers, is significantly stabilized for a long time upon Ca2+/CaM\nbinding (activated state). In accordance with the reviewer’s comments, we clarified in the\nResults section that the concept of dimer pairing has already been reported, and we\n5\nemphasized that our HS-AFM data reveal changes in the lifetime of the kinase domain complex\nbetween the basal and activated states. The text has been revised to reflect these points.\nP.8, L.209\nWhile this kinase domain complex is present in the CaMKIIα subunit, it is more characteristic\nof the CaMKIIβ subunit, as it forms at a higher frequency in the CaMKIIβ subunit\n(Supplementary Fig. 7b, c).\nPrevious studies using single-particle electron microscopy on CaMKIIα21 and\nCaMKIIβ22 homooligomers, crystallographic analysis of the Caenorhabditis elegans CaMKII\n(CeCaMKII) kinase domain17, and various biophysical approaches41,42 have reported the\nformation of kinase domain dimer pairs both in vitro and in vivo43,44. Our HS-AFM observations\nextend these findings by directly visualizing the dynamic behavior of kinase domains, providing\nclear evidence of kinase domain complex formation. Importantly, these complexes exhibit\nhighly dynamic assembly and disassembly patterns, transitioning to a more stable state with\nCa2+/CaM binding, leading to the long-lasting complex state (Fig. 3).\n2. How does the Alphafold3 kinase dimer complex model match the available kinase domain\ndimer structures for camkiia? The authors should take advantage of the rich structural data\nthat is already available (especially on kinase domains) for various camkii isoforms. A related\npoint is that the structural details are not clear in the figure. 4g.\nReply)\nThank you for this constructive suggestion. As the reviewer noted, multiple basal-state CaMKII\nkinase domain structures have been reported, including the activated state of the human\nCaMKIIδ monomer (PDB: 2WEL).\nFor comparison at the monomer level, we aligned the AF3-predicted CaMKIIβ monomer\nwith the CaMKIIδ/Ca2+/CaM crystal structure. Quantitative analysis of Cα positions revealed\ndeviations ≤ 3 Å, indicating robust structural similarity between the two activated-state\nmonomers (Supplementary Fig. 7e).\nFor comparison at the dimer level, we performed protein–protein docking using the\nCaMKIIδ/Ca2+/CaM complex with ClusPro. The docking predicted a stable dimer in which the\ncatalytic sites face inward, thereby shielding the activation site (K43), while still leaving access\nto the T-site surface (Supplementary Fig. 7f). This configuration is in good agreement with our\nAF3-predicted CaMKIIβ dimer model.\nTaken together, these analyses demonstrate that the AF3-predicted activated-state dimer\nof CaMKIIβ, although derived from a different isoform, is consistent with experimentally\nobserved features of CaMKII kinase domain activation. To clarify this point, we have added\n6\nthe comparison results to Supplementary Fig. 7d and expanded the explanation of Fig. 4i in\nthe revised manuscript.\nP.11, L.314\nTo date, the activated state of the CaMKII kinase domain has been crystallographically\ncharacterized only for the human CaMKIIδ monomer (CaMKIIδ/Ca2+/CaM complex, truncated\nCaMKIIδ residues 1–333; PDB: 2WEL)41. First, we used AF3 to predict the structure of the\nactivated state of the CaMKIIβ kinase domain (residues 1–287, excluding the autoinhibitory\nsegment). The predicted structure closely matches the previously reported structure of the\nCaMKIIδ/Ca2+/CaM kinase domain41, supporting that AF3 is capable of accurately modeling\nthe activated state of CaMKII (Supplementary Fig. 7d, e). Furthermore, using this model, we\npredicted the dimeric structure of the activated CaMKIIβ kinase domain. The predicted dimeric\nstructure revealed that one part of the kinase domain covers the active site of the other kinase\ndomain (Fig. 4i). However, the pT287 site was found to be free, adopting a conformation\naccessible to phosphatases.\nAdditionally, we performed protein‒protein docking on the CaMKIIδ/Ca2+/CaM\ncomplex using ClusPro46-50. The docking results suggest a stable dimer in which the catalytic\nsites face inward, effectively masking the essential residue for the catalytic activity (K43), while\ninterstitial gaps allow access to the target binding site (T-site) surface (Supplementary Fig. 7f).\nThis predicted dimer configuration aligns well with our AF3-predicted activated structure of the\nCaMKIIβ dimer.\nFig. 4i\nThe dimer structure conceals K43, which is essential for the catalytic activity of the kinase\ndomain.\nSupplementary Figure 7\nd, Overlaid structure of AF3-predicted CaMKIIβ (residues 11–276) and CaMKIIδ/Ca2+/CaM\nkinase domains.\ne, Cα-Cα distance and root-mean-square deviation (RMSD) of Cα positions between the\nkinase domains of AF3-predicted CaMKIIβ and CaMKIIδ/Ca2+/CaM.\nf, Dimer model derived from protein‒protein docking of the CaMKIIδ/Ca2+/CaM kinase domain.\n3. The western blots in Fig. 4 should be presented with a loading control where both total\nCamKII a/b loaded and associated phospho signals are shown. Currently, no such\nnormalization is presented for these blots.\nReply)\nThank you for the constructive suggestions. In response, we have now included the bands for\ntotal sfGFP-Syn-2 (Fig. 4b) and total CaMKIIα/β (Fig. 4g), as the reviewer recommended.\nHowever, we did not normalize the phosphorylation bands to the corresponding total\nbands. The primary reason is technical: reliable antibodies for CaMKIIα/β are lacking, although\nwe tested several anti-CaMKIIα/β antibodies. In Supplementary Fig. 8, we used an anti-His-\ntag antibody to detect CaMKII. Yet, in the experiments presented in Fig. 4, the samples also\ncontain other His-tagged proteins (PPP2CA, PTPA, and sfGFP-Mini-A), which would overlap\nwith the CaMKII signals and confound quantification.\nWe also note that normalization is most useful for cell-based assays, where the number\nof cells recovered from each dish may vary considerably. In contrast, the present experiments\nemployed purified proteins, where the input amounts are well controlled and such variability\ndoes not occur. Therefore, normalization of phosphorylation signals to total bands would not\nprovide additional benefit in this specific experimental setting.\nTaken together, we have followed the reviewer’s suggestion by presenting the relevant\ntotal bands but avoided potentially misleading normalization. We believe this approach\nprovides the most reliable representation of our phosphorylation assays.\nFig. 4 Caption\nTotal sfGFP-syntide-2 detected by anti-GFP antibody is also presented.\nc, Quantification of the experiments. The band intensity of phosphorylation was measured\ndirectly, without normalization to the Syn-2 (total) band intensity.\ne-h, Representative western blot and quantification of CaMKII dephosphorylation by PP2A.\npT286 (CaMKIIα) or pT287 (CaMKIIβ) was prepared as described in the Methods section, and\nPP2A was added at 0 min. For rat CaMKII, the bands appeared slightly smeared; therefore,\nthe dense lower band was quantified. The phosphorylation band intensity was measured\ndirectly, without normalization to the CaMKII (total) band intensity, as total band intensities\nwere affected by poor antibody recognition. The error bars represent SEMs, and the number\nof experiments is indicated in the figure. NS, not significant (P > 0.05); unpaired t test.\n4. There is no data to show the quality of the purified protein under various conditions. CaMKII\nis notoriously prone to aggregation, especially under conditions of Ca/CaM+ATP treatments.\nSo, a size exclusion profile to show the qualities of the representative protein used under the\nkey conditions will be reassuring.\nReply)\nThank you for the comment. In the basal state, we confirmed the high purity of our samples\nthrough silver staining (Supplementary Fig. 3a). Additionally, under Ca2+/CaM+ATP treatment,\nHS-AFM observations over an area of 300 × 300 nm² revealed dispersed individual CaMKIIβ\nhomooligomers with no signs of aggregation (indicated by white circles in the following figure).\nspecies would have been readily detected; therefore, an additional size exclusion profile is not\nrequired. These results demonstrate that our protein preparations are of high quality with no\naggregation.\nFigure for Reviewers\n5. The beta linker is said to be ~96 residues. The human b linker can be as long as ~200\nresidues. Which splice variant is being used? Uniprot identifiers and variant identifiers will\nimprove data reproducibility. Also, how does changing the linker length from, say, 96 to the b\nvariant that has a 200-residue linker change the dynamics of the kinase domain complexes?\nReply)\nThank you for your constructive comment. As the reviewers mentioned, multiple splicing\nvariants of CaMKIIβ exist, differing in the number of amino acid residues in the linker region.\nBecause our original manuscript did not specify which splicing variant was used, we have\nadded the UniProt ID (P28652) to the Methods section. We selected this variant because it is\nthe most frequently detected CaMKIIβ splice form in humans, as reported previously [Sloutsky\net al. 2020 Sci. Signal. PMID: 32694170].\nRegarding the longer splice variants, certain CaMKIIβ isoforms contain linkers extending\nto ~200 residues. Such extended linkers are expected to influence the spatial flexibility and\ndynamics of the kinase domains, potentially altering their relative positioning within the\nholoenzyme. However, the present study focused on the predominant human variant with a\n~96-residue linker, and our conclusions are based on this physiologically most common form.\nExploring the dynamics of variants with longer linkers remains an important goal for\nfuture research. However, because it slightly diverges from the main focus of our current study,\nwe believe such experiments should be pursued in future work. We appreciate your valuable\nand insightful advice.\nP.16, L.464\n(Addgene plasmid #21227).\n6. Past studies have shown differences in propensity of T286 versus 305/306 phosphorylation\nbetween a and b homooligomers. Can the observed dynamics explain these data? I think it is\ninteresting based on the linker lengths that change the probability of camkiia versus camkiib\nkinases to autophosphorylate in trans versus cis, which can alter the phosphorylation\nprobabilities at the 286/287 versus 305/306 sites. Can the authors please comment on this in\nthe light of their data, where they can see the distances between hub and kinases distinctly for\nboth isoforms?\nReply)\nThank you for your helpful and constructive suggestions. As the reviewers noted, previous\nstudies (CaMKIIβ with a 200 amino acid linker, PMID: 32149607) have shown that\nphosphorylation at T306/T307 (α: T305/T306) occurs more readily than at T287 (α: T286) in\nCaMKIIβ. Our biochemical experiments support this, confirming that pT307 is preferentially\nphosphorylated in CaMKIIβ (Lane 5 in Supplementary Fig. 8).\nFurthermore, a detailed structural comparison of the Ca2+/CaM-bound states of CaMKIIα\nand CaMKIIβ, observed by HS-AFM, revealed that a gap of approximately 6 nm exists between\nthe hub domains and Ca2+/CaM-bound kinase domains only in CaMKIIβ (Supplementary Fig.\n6e). Although it remains unclear whether this extended gap preferentially facilitates cis or trans\nphosphorylation, this increased spatial gap, which is absent in CaMKIIα, may increase the\nlikelihood of either self-phosphorylation (cis) or phosphorylation by neighboring kinase\ndomains (trans). These findings provide new insights that are accessible only through single-\nmolecule imaging by HS-AFM; therefore, we have added the following sentence and\nSupplementary Figure 6e to the revised manuscript. Thank you again for your valuable and\nconstructive suggestions.\nP.9, L.235\nNotably, our biochemical analyses demonstrated that phosphorylation at Thr306\n(β:Thr307) occurs more efficiently in CaMKIIβ, as shown by the stronger signal in Lane 5\ncompared with Lane 2 in Supplementary Figure 8. These findings are consistent with those of\nprevious studies on an alternatively spliced variant of CaMKIIβ containing a longer linker\n(approximately 200 a.a.)20, which also displayed a stronger phosphorylation signal at pT307.\nDetailed structural comparisons of the Ca2+/CaM-bound states of CaMKIIα and CaMKIIβ,\nobserved by HS-AFM, revealed that CaMKIIβ adopts a conformation with an enlarged gap\n(approximately 6 nm) between the hub and Ca2+/CaM-bound kinase domains (Supplementary\nFig. 6e). This increased separation, which is absent in CaMKIIα, likely provides the additional\nconformational space necessary for either cis or trans kinase interactions to reach the Thr307\n10\nphosphorylation site. Such a spatial gap may therefore underlie the preferential\nphosphorylation of Thr307 in CaMKIIβ.\n11"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors provided some explanations to my comments, but concern to all my critiques remain.\n1. Electrostatics in the protein-surface interaction and the effect of the surface charge on the protein dynamics. The authors performed experiments with bare making and detected low mobility of CaMKII on the surface. They comment: “Taken together, we concluded that AFM substrates with weak positive charges (C2P[5]A+) closely approximate physiological dynamics and are optimal for observing the natural behavior of kinase domains”. The conclusion is not clear. If a negatively charged mica surface decrease the dynamics, why a positively changed surface is the right choice? A negative change of the intracellular membrane is a rather common feature of the membrane bilayers. The results they obtain suggest that electrostatic interaction with surface is the factor that contributes to the dynamics of CaMKII in native environment. Experiments with surfaces mimicking the native environment could help them to approach their experimental conditions to the ones in vivo.\n2. Effect of the ionic strength on the protein dynamics. The authors did experiments at 15 mM NaCl with unclear output of the experiments at 150 mM NaCl, which are close to physiological ones. Have they tried imaging on bare mica at high salt conditions? The interactions with the surface can be weaken allowing the authors to visualize the dynamics of CaMKII on negatively charged surface. Just a thought.\n3. Molecular modeling. The authors provided explanation to the difficulty in modeling of such large system, but making simulations was their choice. They refer to EM data, but no clear validation is done. So this section is speculative and needs to be re-written taking into account the lack of the model validation or removed.", "reviewer_2": "The authors have provided satisfactory responses to all my comments and questions, and I strongly recommend publication."}, "author_response": "Response to the reviewers’ comments and remarks\nManuscript ID; NCOMMS-25-35814A\nWe sincerely thank the reviewers for dedicating their valuable time and effort to reviewing\nour paper on two occasions. We appreciate the constructive feedback on our research. We\nbelieve that their contributions and suggestions have enhanced the clarity and quality of the\nmanuscript. Revised parts of the manuscript and answers to each comment included in this\ndocument are in red, and responses to reviewers’ comments are in blue.\nReviewer #1 (Remarks to the Author):\nThe authors provided some explanations to my comments, but concern to all my critiques\nremain.\n1. Electrostatics in the protein-surface interaction and the effect of the surface charge on the\nprotein dynamics. The authors performed experiments with bare making and detected low\nmobility of CaMKII on the surface. They comment: “Taken together, we concluded that AFM\nsubstrates with weak positive charges (C2P[5]A+) closely approximate physiological\ndynamics and are optimal for observing the natural behavior of kinase domains”. The\nconclusion is not clear. If a negatively charged mica surface decrease the dynamics, why a\npositively changed surface is the right choice? A negative change of the intracellular\nmembrane is a rather common feature of the membrane bilayers. The results they obtain\nsuggest that electrostatic interaction with surface is the factor that contributes to the\ndynamics of CaMKII in native environment. Experiments with surfaces mimicking the native\nenvironment could help them to approach their experimental conditions to the ones in vivo.\n2. Effect of the ionic strength on the protein dynamics. The authors did experiments at 15\nmM NaCl with unclear output of the experiments at 150 mM NaCl, which are close to\nphysiological ones. Have they tried imaging on bare mica at high salt conditions? The\ninteractions with the surface can be weaken allowing the authors to visualize the dynamics\nof CaMKII on negatively charged surface. Just a thought.\nReply)\nWe thank the reviewer for these valuable comments and fully agree with the concerns raised.\nBecause comments 1 and 2 address related issues regarding physiological ionic conditions\nand surface properties, we respond to both points together below. We realize that our original\nwording may not have sufficiently clarified the rationale behind our choice of imaging\nconditions, and we appreciate the opportunity to elaborate here.\nAs noted by the reviewer, the impaired dynamics observed on a negatively charged\nmica surface do not necessarily mean that measurements on a positively charged surface\naccurately reflect physiological behavior. We acknowledge this error and correct our previous\nclaim. Below, we explain why we chose the C2P[5]A+ coating and 15 mM KCl as our imaging\nconditions.\nBecause the temporal resolution of HS-AFM is approximately 0.3 s per frame, it is\ninsufficient to capture kinase domain dynamics when the kinase domains are moving\ncompletely freely in solution. Therefore, we needed to establish substrate treatment and\nbuffer conditions that would induce weak interactions between the kinase domains and the\nsubstrate to slow down their movement. After testing various substrate treatments and buffer\nconditions, we found that under the C2P[5]A+ coating and 15 mM KCl buffer, we were able\nto observe CaMKII molecules in which the hub domain is stably attached to the surface, while\nthe kinase domains exhibit dynamic movements involving weak interactions with the\nsubstrate. The weak interaction between the kinase domains and the surface slows down\ntheir movement, enabling us to resolve individual kinase domains with sufficient spatial\nresolution using HS-AFM.\nThus, the expression “approximate physiological dynamics” was misleading. What\nwe actually intended to convey is that the kinase domains are not fully adsorbed onto the\nsubstrate but are able to move to an extent that allows their dynamics to be visualized by\nHS-AFM.\nTo make the things clear. We have added the following sentences.\nP.5, L.117\nBy selecting the AFM substrate and buffer conditions, we are able to observe CaMKII\nmolecules in which the hub domain is stably attached to the AFM substrate, while the kinase\ndomains exhibit dynamic movements involving weak interactions with the AFM substrate.\nAdditionally, regarding the imaging buffer, we performed HS-AFM observations of\nCaMKII on bare mica under conditions mimicking the intracellular environment (15 mM NaCl,\n150 mM KCl and 10 mM MgCl₂, representing a high salt concentration). However, when the\ninteraction between the kinase domain and the AFM substrate is too weak, the movement of\nthe kinase domain is too rapid to be imaged by HS-AFM.\nAlthough intracellular ionic conditions are approximately Na⁺ ~10 mM, K⁺ ~140 mM,\nand Cl⁻ ~5 mM, reproducing these exact conditions in HS-AFM experiments remains\ntechnically challenging. Indeed, at 150 mM KCl concentrations on bare mica, the interaction\nbetween kinase domains and mica surface became too weak, leading to excessively fast\nmovements that could not be captured by HS-AFM. Future developments in HS-AFM with\nhigher temporal resolution may allow us to observe movements more closely mimic the\nnative intercellular environment on unmodified mica surfaces. As these descriptions indicate\nthe limitations of the experiment, we have added the following sentence to the Discussion\nsection of the revised manuscript. Furthermore, to avoid implying that our experimental\nconditions simulate physiological dynamics, the revised manuscript has modified the text as\nfollows.\nP.14, L.401\nOur HS-AFM observations are limited to monitoring the dynamics of the kinase domains\nof CaMKIIα/β in two-dimensional (2D) space on the AFM substrate. Additionally, our\nexperimental conditions–comprising AFM substrate and buffer conditions– were optimized to\nobserve CaMKII movement using HS-AFM; however, the mobility of kinase domains within\nliving cells may be faster. Improvements in the temporal resolution of HS-AFM in the future\nhave potential to capture CaMKII movements under more physiologically relevant conditions.\nP.19, L.576\nWe note that the dynamics at 150 mM KCl looks similar to those at 15 mM KCl. Therefore,\nas described in our previous study39, we conclude that while salt concentration influences\nthe interaction between CaMKII oligomers and the AFM substrate, the conditions used\nin this study—including the substrate and buffer conditions—were optimized to observe\nthe kinase domain dynamics of CaMKII and allow for comprehensive analysis.\n3. Molecular modeling. The authors provided explanation to the difficulty in modeling of such\nlarge system, but making simulations was their choice. They refer to EM data, but no clear\nvalidation is done. So this section is speculative and needs to be re-written taking into\naccount the lack of the model validation or removed.\nReply)\nWe thank the reviewer for this valuable comment. In response to the reviewer’s comment,\nwe have removed these mathematical models from the revised manuscript. In addition,\nFigure 7 has been moved to Supplementary Figure 10. Although Figure 7 (now\nSupplementary Fig. 10) is speculative, we believe that its inclusion in the manuscript provides\nmeaningful value to the reader. In particular, the model offers a conceptual framework to\ninterpret the HS-AFM observations and helps to clarify possible mechanisms underlying\nCaMKII–actin interactions. Additionally, it provides a useful basis for guiding future\nexperimental studies aimed at quantitatively validating and refining these mechanisms. For\nthese reasons, we consider it appropriate to retain the model in the Supplementary\nInformation and to discuss it clearly within the revised manuscript.\nP.13, L.384, L.385\n(Supplementary Fig. 10).\nP.14, L.400\n(Supplementary Fig. 10b)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have provided satisfactory responses to all my comments."}, "author_response": ""}]

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