Ethan_McSpadden
Ethan Stamp


Variation in assembly stoichiometry in non-metazoan homologs of the hub domain of Ca2+/Calmodulin-dependent protein kinase II

Ethan D. McSpadden, Zijie Xia, Chris C. Chi, Anna C. Susa, Neel H. Shah, Christine L. Gee, Evan R. Williams and John Kuriyan

Protein Science 2019 28:1071–1082     (local copy)

BioRχiv 536094; doi: https://doi.org/10.1101/536094

Abstract

The multi-subunit Ca2+/calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C-terminal hub domain that assembles into a 12- or 14-subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII-like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16-, 18-, and 20-subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from Chlamydomonas reinhardtii reveals an 18-subunit organization. We identified four intra-subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII-α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12- and 14-subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.

Figures from the paper

(Click on the small image to get a higher-resolution version.)
Figure 1 from paper

Figure 1 - CaMKII architecture.


A. Domain organization for an intact CaMKII subunit.
B. Depiction of the CaMKII holoenzyme, C-terminal hub domains (shown in gray) oligomerize into a donut shaped structure, forming the core of the holoenzyme. N-terminal kinase domains (shown in blue) extend outwards from the core, tethered to the hub by a flexible linker. The holoenzyme depicted here is a dodecamer, but tetradecameric holoenzymes can be formed as well (Bhattacharyya et al. 2016; Myers et al. 2017) . In this diagram, which represents the activated state of CamKII, the autoinhibitory regulatory segments (yellow) are shown displaced from the kinase domains.
C. A phylogenetic tree of some organisms that encode forms of CaMKII. Full length CaMKII is only found in metazoans and premetazoan choanoflagellates. Isolated CaMKII hub domains are found in the genomes of some green algae and some bacteria.

Figure 2 from paper

Figure 2 - Sequence alignment of CaMKII hub domains.


Sequence alignment of the human CaMKII-α hub domain and the hub domains encoded by C. elegans, N. vectensis, S. rosetta, Chlamydomonas, Volvox, Gonium and Pirellula. The human to Chlamydomonas point mutation sites that shifted the preferred oligomerization states of the human hub domain are marked with green diamonds. The residues that are buried in the Chlamydomonas and mutant human hub domain structures are marked with a blue circle. Yellow indicates high sequence identity and grey represents partial sequence identity.

Figure 3 from paper

Figure 3 - Mass spectra of hub complexes.


A. Electrospray ionization mass spectrum (ESI-MS) under native conditions of the Chlamydomonas CaMKII hub domain. A single species was detected with a molecular weight of 309,105 Da, corresponding to an 18-subunit assembly.
B. ESI-MS of the Volvox CaMKII hub domain. Two species were detected with molecular weights of 275,261 and 305,798 Da, corresponding to 18-subunit and 20-subunit assemblies, respectively.
C. ESI-MS of the Gonium CaMKII hub domain. Two species were detected with molecular weights of 275,440 and 309,860 Da, corresponding to 16-subunit and 18-subunit assemblies, respectively.
Figure 4 from paper

Figure 4 - Comparison of the Chlamydomonas and human hub domain structures.


A. Chlamydomonas and human domains have a highly conserved structure, with a large N-terminal helix cradled by a highly curved antiparallel β-sheet. The conserved arginines in an internal cavity are circled.
B. The structure of the hub domain vertical dimer is highly conserved between the human and Chlamydomonas proteins.

Figure 5 from paper

Figure 5 - The Chlamydomonas CaMKII hub domain assembly.


A. The Chlamydomonas CaMKII hub domain forms an 18-subunit assembly. The protomers are arranged as nine vertical dimers arranged side-by-side. The upper edge of the β-sheet of each protomer is colored in orange to help delineate the subunits. A vertical dimer of protomers (Stratton et al. 2014) is also highlighted in teal for clarity.
B. The dodecameric human CaMKII hub domain assembly is presented for reference. The β-sheet edges are rendered in pink and a vertical dimer is highlighted in purple.

Figure 6 from paper

Figure 6 - Comparison of the Chlamydomonas, S.Rosetta, mouse and human hub domain structures.


A single hub domain from each of open spiral S. Rosetta (5ig0; pink), human 12-subunit (5ig3; grey), mouse 14-subunit (1hkx ; blue), and Chlamydamonas 18-subunit (green) assemblies were aligned along the central helix and the right most β-sheet section in this view (residues 344-380, 418-430 and 451-469 in the human hub domain). There is a progressive increase in curvature of the β-sheet with assembly size.

Figure 7 from paper

Figure 7 - Additional intra-chain hydrogen bonds.


A. Gln 12 makes an interaction with the backbone carbonyl of Asn 74. The human sequence has a glutamate at this position which is unable to form the interaction.
B. Hydrogen bond between Asn 74 and Arg 95. A threonine residue is present at this location in the human alpha isoform and it is not positioned to form a hydrogen bond with the conserved arginine residue.
C. Hydrogen bond between His 125 and Arg 114. The histidine is replaced by an isoleucine in the human isoform.
D. Asn 11 forms a hydrogen bond with Trp 116 and also potentially a hydrogen bond with Tyr 93. Asn 11 is replaced by a threonine in the human isoform that forms a hydrogen bond with the tryptophan but cannot simultaneously interact with the tyrosine.

Figure 8 from paper

Figure 8 - Oligomerization states of Mutant and wild-type human hub domains.


A.Oligomerization states of a mutant human CaMKII-α hub domain. Only 14-subunit and 16-subunit assemblies are formed.
B. Oligomerization stats of the wild-type human CaMKII-α hub domain. Only 12-subunit and 14-subunit assemblies are formed, in roughly equal proportions.

Note: 14-subunit m/z values differ between mutant and wild-type because the expression tag was not cleaved prior to acquisition of the wild-type spectrum.


Supplemental figures from the paper

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Supplemental Figure 1 from paper

Supplementary Figure 1


A. Electrosprayionization mass spectrum (ESI-MS) of the CaMKII hub domain encoded by the bacterium Pirellula sp. SH-Sr6A (UniprotKB AMV33243.1). Two species were detected with molecular weights of 207,866 ± 266 and 60,144 ± 498 Da, corresponding to tetradecamers (14-subunits) and tetramers (4-subunits). The mass spectrum was obtained at 91 μM subunit concentration.
B. ESI-MS of the Pirellula sp. hub domain in in 2.5 mM Tris, 15 mM KCl, and 1% glycerol solution with ~1000-fold lower protein concentration. The 14-subunit oligomer is still formed, although the abundance of dimer and tetramer are higher.
C. Tandem mass spectrum of the CamKII hub domain from Chlamydomonas showing two monomer masses of 17090 ± 2 and 17268 ± 6 Da. The former matches the sequence mass. The ratio of the lower mass to larger mass monomer is approximately 2 to 1.

Supplemental Figure 2 from paper

Supplementary Figure 2


A. Crystal structure of the mutant human CaMKII-α hub domain. The protein crystallized in tetradecameric form. The overall structure is generally the same as the wild-type tetradecamer (PDB 1HKX), shown in B. as a reference.

Supplemental Figure 3 from paper

Supplementary Figure 3


A. Left panel: Interaction between mutant Gln 355 and backbone carbonyl 412 (blue structure). The glutamine is positioned to form a hydrogen bond with the carbonyl, but the distance is longer than a typical hydrogen bond at 3.6 Å. The wild-type structure (PDB 1HKX, shown in grey) is aligned on helix α1 for reference. Note that the backbone carbonyl of the mutant structure is closer to the helix, indicating that the β-sheet is slightly more curved relative to wild-type. Right panel: The glutamine sidechain-backbone carbonyl hydrogen bond in the Chlamydomonas CaMKII hub domain.
B. Left panel: Hydrogen bond between mutant Asn 412 and conserved Arg 433 (blue structure). The wild-type structure (in grey) is again aligned on helix α1 for reference. The wild-type threonine residue is not positioned to form this hydrogen bond. Right panel: The asparagine-conserved arginine hydrogen bond in the Chlamydomonas structure.

Supplemental Figure 3 from paper

Supplementary Figure 3 continued


C. Left panel: Hydrogen bond between mutant His 464 and conserved Arg 453 (blue structure). Isoleucine is the wild-type residue at position 464 (grey structure). Right panel: The histidine-conserved arginine hydrogen bond in the Chlamydomonas structure.
D. Left panel: Hydrogen bonds between mutant Asn 354 and conserved residues Trp 455 and Tyr 431. In the wild-type structure (shown in grey) Thr 354 forms a hydrogen bond with the tryptophan but cannot simultaneously interact with the tyrosine. Right panel: Hydrogen bonds formed between Asn 33, Trp 138, and Tyr 115 in the Chlamydomonas CaMKII hub domain.