Conformational spread drives the evolution of the calcium-calmodulin protein kinase II

Abstract

The calcium calmodulin (Ca²⁺/CaM) dependent protein kinase II (CaMKII) decodes Ca²⁺ frequency oscillations. The CaMKIIα isoform is predominantly expressed in the brain and has a central role in learning. I matched residue and organismal evolution with collective motions deduced from the atomic structure of the human CaMKIIα holoenzyme to learn how its ring architecture abets function. Protein dynamic simulations showed its peripheral kinase domains (KDs) are conformationally coupled via lateral spread along the central hub. The underlying β-sheet motions in the hub or association domain (AD) were deconvolved into dynamic couplings based on mutual information. They mapped onto a coevolved residue network to partition the AD into two distinct sectors. A second, energetically stressed sector was added to ancient bacterial enzyme dimers for assembly of the ringed hub. The continued evolution of the holoenzyme after AD-KD fusion targeted the sector's ring contacts coupled to the KD. Among isoforms, the α isoform emerged last and, it alone, mutated rapidly after the poikilotherm-homeotherm jump to match the evolution of memory. The correlation between dynamics and evolution of the CaMKII AD argues single residue substitutions fine-tune hub conformational spread. The fine-tuning could increase CaMKIIα Ca²⁺ frequency response range for complex learning functions.

Figure 1

(a) Architecture. (i) Subunit. Disordered linkers with varying lengths and composition connect the kinase domain (KD (N-lobe (orange), C-lobe (white)) with the association domain (AD). The pseudo-substrate, regulatory segment (R (brown) binds Ca²⁺/CaM. The AD β-sheet forms vertical (red) and lateral (magenta) ring contacts. (ii) Assembly. The ADs form the central hub in the multi-subunit holoenzyme (CaMKIIα 3SOA.PDB). A tetramer (circle) was extracted for analysis of conformational fluctuations. (b) Flexure. The flexibility (rmsf)) profile derived from the tetramer conformational ensemble. The tetramer orientation shown is rotated by 90° relative to the orientation in panel a (Supplementary Video S1). (c) Principal Component Analysis (PCA). (i). PCIPC2PC3 plots of the human CaMKIIα AD; (ii) A conformation in the CaMKIIα tetramer tCONCOORD ensemble (Supplementary Video S2) used for PCA, with PCs 1–3 mapped onto the key contacts (AD Vertical (Vert-Dim), Tilt. AD Lateral (Lat-Dim), Bend and twist. KD–AD. Crankshaft (extension + rotation)). Rectangles represent β sheet long axes. (d) Monomer network. Nodes = Residues (Circles). Edges = Dynamic couplings between 4-residue fragments (lines color-coded according to nMI score (high (orange) → low (blue)). Source listed in Table S1.

Figure 2

The dynamics and evolution of the CaMKII AD. Hub interfaces and R helix are color-coded as in Fig. 1a. (a,b). KD–AD Contact. (a). Dynamics. The top dynamic couplings computed between 4-residue fragments (yellow (weak) → orange → red (strong)). The KD–AD contact residues and surface (yellow). (b) Energetics. Energy frustration—(relaxed (green), stressed (red)). Spheres denote T286 (red), T305 (cyan). (c,d) AD Fold. (c) Interfacial dynamics. ACGI AD tetramer. The dynamic couplings span the interfaces (Vert-Dim (red), Lat-Dim (purple). Supplementary Video S3). (d) Energetics. Energy frustration scores are color coded as in B. (e) Residue Coevolution. The superposition of the dynamic (thin orange lines) and coevolved (thick salmon lines) couplings adjacent to the (i) Vert-Dim and (ii) Lat-Dim contacts (stick sidechains). 3D-views in Supplementary Videos S4–S7. Source listed in Table S1.

Figure 3

The two sectors of the CaMKIIα AD. The coevolved residue network of the primordial vertical dimer (Sector V) has energetically relaxed residues (green spheres) at the Vert-Dim contact interface (red β strands). The coevolved residue network (thick salmon lines) of the Lat-Dim contact (Sector L) has energetically stressed residues (red spheres) at the contact interface (purple α-helix, β strands). Stick representations denote residues at the interface (purple, green, red) or part of adjacent dynamic couplings (gold). 3D-view in Supplementary Video S8. Source listed in Table S1.

Figure 4

CaMKII phylogenetics (from 3D Structures). (a). Tree of 23 crystal structures of the CaMKII-AD homologs (color-coded by phyla) based on DALI scores. (b). The evolutionary trace (EV trace) for structures of an ancient bacterial enzyme (40VM.PDB) versus human CaMKIIα (3SOA.PDB). 3D Views in Supplementary Videos S9–S10. (C). Conservation of the dimer contact (RMSD = 0.69 nm). Source listed in Table S1.

Figure 5

CaMKII phylogenetics (from 1D Sequences). (a). Metazoan CaMKII-AD evolution. Tree based on 1000 clustered homologs of the C. elegans CaMKII (black asterisk). Midge (pink asterisk). (b). Isoform evolution. Tree based on 2000 clustered homologs of the rat CaMKII α and β isoforms. Squares denote the most distant, larger CaMKIIα node (L) and the smallest y node with H:P ratios > $\overline{\text{H}:\text{P}}$ ± 2 s (1.5 ± 1.33), where $\overline{\text{H}:\text{P}}$ is the mean of the other nodes. Circles mark major clusters (diameter = membership; color = (i) phylum; (ii) isoform), Note difference in scale bar from that in Fig. S2C. (c) AD–KD coupling. The AD versus KD similarity plot compared the KD and AD phylogenetic trees color coded by isoform as in panel b (Box) The dual color symbols identify isoform pairs. The correlation coefficient, r = 0.59 with the best-fit (solid line) ± 95% confidence intervals (dashed lines). (d). Evolution of memory. Phylogenetic tree based on behavioral assays (from). Arrows show the major bifurcations associated with the emergence of homeotherms (blue) and primates (red). Box: The distribution of the poikilotherms (blue, cyan) and homeotherms (rouge, red, orange, salmon) kingdoms in the sequences of the δ isoform. (e). Phylogenetic Species Diversity: Species distributions for major nodes of the α, β and γ isoforms based on kingdom (i) and homeostasis (ii). Source listed in Table S1.

Figure 6

The correlation between the structural evolution of CaMKII and sensory behavior. Structure. The selection of the dimer (D) from other structures (M = monomer, T = tetramer) in bacteria seeded the emergence of ringed hub assemblies. The fusion with the kinase (K) domains coincided with the emergence of multicellularity (secondary structures color coded as in Fig. 1). Diversity was created by linker alternative splicing and enhanced by the generation of isoforms. Behavior. Work on model organisms suggests CaMKII evolution peaks with the development of cognitive memory. Advanced memory mechanisms (orange block). Source listed in Table S1.