Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II
Oren S. Rosenberg, Sebastian Deindl, Luis R. Comolli, André Hoelz, Kenneth H. Downing, Angus C. Nairn and John Kuriyan
Summary: Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C-terminal region of ≈ 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKII? has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. In this study, we have determined the crystal structure of the Caenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal ≈ 1-335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form.
Figure 1: The domain structure of the CaMKII proteins. All isoforms have the same basic architecture although the different isoforms have variable insertions of between 21 and 178 residues in the linker between the kinase and the association domain.
Figure 2: Static light scattering analysis of M. musculus CaMKII holoenyzme. (A) Laser light scattered in a single direction from the eluant of a gel filtration column. Signal was measured at 0.5 s intervals as a function of elution volume (red line, reported in the primary units of the signal, Volts). The relative concentration of protein, as measured by the refractive index, is also shown (blue). (B) A plot of the molar mass predicted from the analysis of the concentration and scattering data as a function of elution volume (red dots). Superimposed for reference is the same concentration curve shown above (blue line). The area highlighted in yellow is the portion of the concentration and scattering curves used in the analysis.
Fiugre 3: Uranyl acetate stained images of the M. musculus holoenyzme reveal a 6-fold symmetry. (A) Electron microscopic images and class averages. (i) Three representative raw images of single particles. (ii) Six representative class averages, all of which look very similar, suggesting a limited distribution of orientations on the grid. (B) The first four eigen images of the association domain classification. The first eigen image shows a strong 6-fold modulation, as seen in the inset expanded view.
Figure 4: Cryo-electronmicroscopy reveals the position of the kinase domains of CaMKII. (A) A representative class average (1 of 5) from the C. elegans holoenyzme micrographs. The central ring of density of ≈ 10 nm radius is presumed to be the association domain. The outer ring of density of ≈ 22 nm radius is presumed to be the kinase domains. (B) As in (A) but from the mouse CaMKIIα isoform micrographs. (C) The first eigen image of the mouse CaMKIIα isoform dataset.
Figure 5: The CaMKIIα association domain (residue 335-478) expressed in bacteria forms 7-fold rings. (A) Electron microscopic images and class averages. (i) Three representative raw images from the micrographs of the bacterially expressed CaMKIIα association domain. (ii) Six representative class averages that look very similar, suggesting a limited distribution of orientations on the grid. (B) The first four eigen images of the association domain classification. The first eigen image shows a strong 7-fold modulation.
Figure 6: Proteolysis of the mouse CaMKIIα holoenzyme leads to a 7-fold symmetric association domain structure. (A) Proteolysis of the M. musculus holoenyzme with trypsin leads to the production of two bands. Subsequent mass spectrometric analysis showed these two bands to be the kinase domain and the association domain as indicated in the figure. (B) Crystallization trials with the mixture shown in (A) produces large diamond like crystals. (C) Molecular replacement with the dimer of association domains reveal a 7-fold ring structure in the electron density of the crystals produced from the proteolyzed material.
Figure 7: Proteolysed C. elegans CaMKII also crystallizes as 7-fold association domain rings. (A) The self rotation function reveals seven 2-fold axes of rotation in the association domain crystals, although one of the 2-fold axes coincides with a crystallographic 2-fold axis. (B,C) The structure of the monomer (B) and the 7-fold association domain ring (C) produced from the proteolysis of the C. elegans CaMKII holoenzyme.
Figure 8: Models of holoenzyme. In the holoenzyme the kinase domains may constrain the ring so as to maintain a dodecameric assembly (A). When the kinase domains are absent this constraint is released allowing the ring to relax into the 7-fold symmetric assembly (B). (C) A 6-fold symmetric association domain model in contrast with (D), the 7-fold association domain crystal structure from proteolyzed C. elegans full-length CaMKII.