Spontaneous non-canonical assembly of CcmK hexameric components from β-carboxysome shells of cyanobacteria

Abstract

CcmK proteins are major constituents of icosahedral shells of β-carboxysomes, a bacterial microcompartment that plays a key role for CO2 fixation in nature. Supported by the characterization of bidimensional (2D) layers of packed CcmK hexamers in crystal and electron microscopy structures, CcmK are assumed to be the major components of icosahedral flat facets. Here, we reassessed the validity of this model by studying CcmK isoforms from Synechocystis sp. PCC6803. Native mass spectrometry studies confirmed that CcmK are hexamers in solution. Interestingly, potential pre-assembled intermediates were also detected with CcmK2. Atomic-force microscopy (AFM) imaging under quasi-physiological conditions confirmed the formation of canonical flat sheets with CcmK4. Conversely, CcmK2 formed both canonical and striped-patterned patches, while CcmK1 assembled into remarkable supra-hexameric curved honeycomb-like mosaics. Mutational studies ascribed the propensity of CcmK1 to form round assemblies to a combination of two features shared by at least one CcmK isoform in most β-cyanobacteria: a displacement of an α helical portion towards the hexamer edge, where a potential phosphate binding funnel forms between packed hexamers, and the presence of a short C-terminal extension in CcmK1. All-atom molecular dynamics supported a contribution of phosphate molecules sandwiched between hexamers to bend CcmK1 assemblies. Formation of supra-hexameric curved structures could be reproduced in coarse-grained simulations, provided that adhesion forces to the support were weak. Apart from uncovering unprecedented CcmK self-assembly features, our data suggest the possibility that transitions between curved and flat assemblies, following cargo maturation, could be important for the biogenesis of β-carboxysomes, possibly also of other BMC.

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

“…Despite formation of 2D patches noticed in some experiments for K3/K4, the assemblies were smaller and less regular than those obtained with His4-tagged K4 homohexamers ( Fig. 5A, right), which basically reproduced previous published results [23]. Assembly plane with K3/K4 samples positioned about 2.8 nm above mica surfaces, though this value is likely an underestimate induced by high surface coverages.…”

“…The proposition was based on the characterization by size-exclusion chromatography of K3/K4 species with apparent size expected for dodecamers (supposedly being double stacked hexamers), and on the assumption that the assembly of K3 paralogs must be hampered by the replacement of two residues that are key in holding inter-hexamer contacts with all other CcmK, and generally across BMC-H. Actually, AFM data presented here demonstrated a decline in assembly tendency of Syn6803 K3/K4 and more surprisingly of the K1/K2 hetero-hexamersresidues implicated in hexamer contacts are identical in K1 and K2, thus suggesting that the same overall effect might be elicited by different means (e.g. the different tendency of each paralog to form curved assemblies [23]) -. However, although K1/K2 displayed in solution the hexamerdodecamer equilibrium characteristic of the K2 paralog, the Syn6803 K3/K4 was hexameric, something that fits with a replacement of E98 by Ala in Syn6803 K3.…”

“…For K1-His4/K2-FLAG, the presence of the K2 paralog was directly evidenced by its two typical charge state distributions (CSD) at m/z 4000-5000 and 5500-6200 m/z. The second CSD, however, corresponded to species of lower MW than those detected before for the K2 homo-hexamer [23]. A zoomed view of the hexamer distribution indicated that every charge state was in fact split into several signals, matching to hexamers of different stoichiometries (shown in the inset of Fig.…”

“…Finally, samples were inspected by native electrospray ionization mass spectrometry (native ESI-MS), an approach that is well suited for the characterization of molecular associations present in solution. This technique was exploited in a previous study to characterize His4-tagged CcmK homohexamers [23]. After improving the molecular homogeneity of TALON-purified fractions by TEV treatment, hetero-hexamers could be detected in the 3500-5000 m/z range by native ESI-MS ( Fig.…”

“…The first one (Pfam00936) is present in proteins that are organized as hexamers (BMC-H) [13][14][15][16][17][18], and as tandem domains in subunits that form trimers (BMC-T) [18][19][20]. These proteins often give rise within crystals to stacked twodimensional (2D) layers, a property that was also confirmed by electron microscopy and atomic force microscopy [21][22][23]. Consequently, these proteins were proposed early to compose the planar facets of BMC shells, which according to various electron microscopy studies, could be polyhedral/icosahedral [24][25][26].…”