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

PLoS One. 2017 Sep 21;12(9):e0185109. doi: 10.1371/journal.pone.0185109. eCollection 2017.

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.

MeSH terms

  • Aluminum Silicates / chemistry
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Chromatography, Gel
  • Isomerism
  • Mass Spectrometry
  • Microscopy, Atomic Force
  • Molecular Dynamics Simulation
  • Mutation
  • Phosphates / chemistry
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Solutions
  • Solvents / chemistry
  • Synechocystis

Substances

  • Aluminum Silicates
  • Bacterial Proteins
  • CcmK protein, Synechocystis sp.
  • Phosphates
  • Solutions
  • Solvents
  • mica

Grants and funding

LFGA, NA, DP and GT work was financed in part within the CarboYeast precompetitive project by Toulouse White Biotechnology. AFM work by EL, NP and EB was founded by ANR-15-IDEX-03 PIA2/iSite-BFC, ANR-15-CE09-0002-02 and Feder grants. KR, KB and RZ acknowledge financial support for mass spectrometry studies by the Swiss National Science Foundation (SNF, grant number 200020_159929). CIM received financial support provided by DGAPA-UNAM through a Sabbatical Fellowship and by grant DGAPA IN-110516. DR acknowledges funding from the Spanish government through grant FIS2015-67837-P (MINECO/FEDER, UE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.