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.
Highlights
Bacterial microcompartments (BMC) are proteinaceous organelles that sequester enzymes that catalyze processes involving toxic or volatile intermediates in a wide variety of bacteria [1]
We provide evidence suggesting that BMC-H components could introduce curvature in BMC shells, something that could be of significance for BMC biogenesis/assembly
Multiple bands were apparent for the K4-TH construct, suggesting sensitivity to cellular proteases, something confirmed by the presence of multiple peaks detected by mass spectrometry (MS) on purified samples
Summary
Bacterial microcompartments (BMC) are proteinaceous organelles that sequester enzymes that catalyze processes involving toxic or volatile intermediates in a wide variety of bacteria [1]. CB is a BMC subtype that participates in a carbonconcentrating mechanism (Ccm) that allows photosynthetic cyanobacteria and chemoautotrophs to enhance carbon fixation yields [3]. CB confine within their shells two key enzymatic activities: ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) and carbonic anhydrases (CA). Apart from shell and mentioned enzymatic contents, CcmM and CcmN proteins of β-cyanobacteria provide scaffolding functions permitting to organize together cargo and shell of their β-CB. Short peptide sequences of scaffolding/ cargo proteins are known to mediate contacts with the shell [10, 11], something that allowed to engineer new BMC with reprogrammed contents [10, 12,13,14]. Other studies evidenced that BMC shell formation might be uncoupled from cargo content[14, 17, 18], a possibility that should not be fully ruled out for β-CB[19]
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