Photosynthesis Research

, Volume 121, Issue 2–3, pp 125–133 | Cite as

Interactions and structural variability of β-carboxysomal shell protein CcmL

  • Thomas J. Keeling
  • Bożena Samborska
  • Ryan W. Demers
  • Matthew S. KimberEmail author
Regular Paper


CcmL is a small, pentameric protein that is argued to fill the vertices of β-carboxysomal shell. Here we report the structures of two CcmL orthologs, those from Nostoc sp. PCC 7120 and Thermosynechococcus elongatus BP-1. These structures broadly resemble those previously reported for other strains. However, the Nostoc CcmL structure shows an interesting pattern of behavior where two loops that map to the base of the pentamer adopt either an out or in conformation, with a consistent (over six pentamers) out–in–out–in–in pattern of protomers. The pentamers in this structure are also consistently organized into a back-to-back decamer, though evidence suggests that this is likely not present in solution. Förster resonance energy transfer experiments were able to show a weak interaction between CcmL and CcmK2 when CcmK2 was present at >100 μM. Since CcmK2 forms defined bodies with approximately 200 nm diameter at this concentration, this would support the idea that CcmL can only interact with CcmK2 at rare defect points in the growing shell.


Carboxysome Microcompartment Cyanobacteria Protein structure FRET Carbon-concentrating mechanisms 



The authors wish to thank Dr. Cezar Khursigara and Elyse Roach for help with the electron microscopy. NpCcmL data were collected at CLS by Shaun Labiuk and Pawel Grochulski. This work was funded by a Discovery Grant from the National Science and Engineering Research Council of Canada to MSK (# 327280).

Supplementary material

11120_2014_9973_MOESM1_ESM.docx (1.1 mb)
Supplementary material 1 (DOCX 1134 kb)


  1. Adams PD, Grosse-Kunstleve RW, Hung L-W et al (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58:1948–1954. doi: 10.1107/S0907444902016657 PubMedCrossRefGoogle Scholar
  2. Cai F, Menon BB, Cannon GC et al (2009) The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier. PLoS ONE 4:e7521. doi: 10.1371/journal.pone.0007521 PubMedCentralPubMedCrossRefGoogle Scholar
  3. Cai F, Sutter M, Cameron JC et al (2013) The structure of CcmP, a tandem bacterial microcompartment domain protein from the β-carboxysome, forms a subcompartment within a microcompartment. J Biol Chem 288:16055–16063. doi: 10.1074/jbc.M113.456897 PubMedCentralPubMedCrossRefGoogle Scholar
  4. Chen AH, Robinson-Mosher A, Savage DF et al (2013) The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo. PLoS ONE 8:e76127. doi: 10.1371/journal.pone.0076127 PubMedCentralPubMedCrossRefGoogle Scholar
  5. Cheng S, Liu Y, Crowley CS et al (2008) Bacterial microcompartments: their properties and paradoxes. BioEssays 30:1084–1095. doi: 10.1002/bies.20830 PubMedCentralPubMedCrossRefGoogle Scholar
  6. Choudhary S, Quin MB, Sanders MA et al (2012) Engineered protein nano-compartments for targeted enzyme localization. PLoS ONE 7:e33342. doi: 10.1371/journal.pone.0033342 PubMedCentralPubMedCrossRefGoogle Scholar
  7. Craciun S, Balskus EP (2012) Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci USA 109:21307–21312. doi: 10.1073/pnas.1215689109 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132. doi: 10.1107/S0907444904019158 PubMedCrossRefGoogle Scholar
  9. Espie GS, Kimber MS (2011) Carboxysomes: cyanobacterial RubisCO comes in small packages. Photosynth Res 109:7–20. doi: 10.1007/s11120-011-9656-y PubMedCrossRefGoogle Scholar
  10. Forouhar F, Kuzin A, Seetharaman J et al (2007) Functional insights from structural genomics. J Struct Funct Genomics 8:37–44. doi: 10.1007/s10969-007-9018-3 PubMedCrossRefGoogle Scholar
  11. Fukuzawa H, Suzuki E, Komukai Y, Miyachi S (1992) A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci USA 89:4437–4441PubMedCentralPubMedCrossRefGoogle Scholar
  12. Held M, Quin MB, Schmidt-Dannert C (2013) Eut bacterial microcompartments: insights into their function, structure, and bioengineering applications. J Mol Microbiol Biotechnol 23:308–320. doi: 10.1159/000351343 PubMedCrossRefGoogle Scholar
  13. Heldt D, Frank S, Seyedarabi A et al (2009) Structure of a trimeric bacterial microcompartment shell protein, EtuB, associated with ethanol utilization in Clostridium kluyveri. Biochem J 423:8883–8887. doi: 10.1073/pnas.0902324106 CrossRefGoogle Scholar
  14. Jorda J, Lopez D, Wheatley NM, Yeates TO (2013) Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria. Protein Sci 22:179–195. doi: 10.1002/pro.2196 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66:125–132. doi: 10.1107/S0907444909047337 PubMedCentralPubMedCrossRefGoogle Scholar
  16. Kerfeld CA, Sawaya MR, Tanaka S et al (2005) Protein structures forming the shell of primitive bacterial organelles. Science 309:936–938. doi: 10.1126/science.1113397 PubMedCrossRefGoogle Scholar
  17. Kerfeld CA, Heinhorst S, Cannon GC (2010) Bacterial microcompartments. Annu Rev Microbiol 64:391–408. doi: 10.1146/annurev.micro.112408.134211 PubMedCrossRefGoogle Scholar
  18. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797. doi: 10.1016/j.jmb.2007.05.022 PubMedCrossRefGoogle Scholar
  19. Long BM, Badger MR, Whitney SM, Price GD (2007) Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA. J Biol Chem 282:29323–29335. doi: 10.1074/jbc.M703896200 PubMedCrossRefGoogle Scholar
  20. Menon BB, Dou Z, Heinhorst S et al (2008) Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species. PLoS ONE 3:e3570. doi: 10.1371/journal.pone.0003570 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: the integration of data reduction and structure solution: from diffraction images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr 62:859–866. doi: 10.1107/S0907444906019949 PubMedCrossRefGoogle Scholar
  22. Palmier MO, Van Doren SR (2007) Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. Anal Biochem 371:43–51. doi: 10.1016/j.ab.2007.07.008 PubMedCentralPubMedCrossRefGoogle Scholar
  23. Parsons JB, Dinesh SD, Deery E et al (2008) Biochemical and structural insights into bacterial organelle form and biogenesis. J Biol Chem 283:14366–14375. doi: 10.1074/jbc.M709214200 PubMedCrossRefGoogle Scholar
  24. Parsons JB, Frank S, Bhella D et al (2010) Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement. Mol Cell 38:305–315. doi: 10.1016/j.molcel.2010.04.008 PubMedCrossRefGoogle Scholar
  25. Peña KL, Castel SE, de Araujo C et al (2010) Structural basis of the oxidative activation of the carboxysomal gamma-carbonic anhydrase, CcmM. Proc Natl Acad Sci USA 107:2455–2460. doi: 10.1073/pnas.0910866107 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Price GD (2011) Inorganic carbon transporters of the cyanobacterial CO2 concentrating mechanism. Photosynth Res 109:47–57. doi: 10.1007/s11120-010-9608-y PubMedCrossRefGoogle Scholar
  27. Price GD, Badger MR (1991) Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism. Can J Bot 69:963–973CrossRefGoogle Scholar
  28. Price GD, Howitt SM, Harrison K, Badger MR (1993) Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function. J Bacteriol 175:2871–2879PubMedCentralPubMedGoogle Scholar
  29. Rae BD, Long BM, Badger MR, Price GD (2012) Structural determinants of the outer shell of β-carboxysomes in Synechococcus elongatus PCC 7942: roles for Ccm K2, K3–K4, CcmO, and CcmL. PLoS ONE 7:e43871. doi: 10.1371/journal.pone.0043871 PubMedCentralPubMedCrossRefGoogle Scholar
  30. Rae BD, Long BM, Badger MR, Price GD (2013) Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77:357–379. doi: 10.1128/MMBR.00061-12 PubMedCrossRefGoogle Scholar
  31. Samborska B, Kimber MS (2012) A dodecameric CcmK2 structure suggests β-carboxysomal shell facets have a double-layered organization. Structure 20:1353–1362. doi: 10.1016/j.str.2012.05.013 PubMedCrossRefGoogle Scholar
  32. So AKC, Espie GS, Williams EB et al (2004) A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell. J Bacteriol 186:623–630PubMedCentralPubMedCrossRefGoogle Scholar
  33. Starcher B (2001) A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal Biochem 292:125–129. doi: 10.1006/abio.2001.5050 PubMedCrossRefGoogle Scholar
  34. Sutter M, Wilson SC, Deutsch S, Kerfeld CA (2013) Two new high-resolution crystal structures of carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species. Photosynth Res. doi: 10.1007/s11120-013-9909-z PubMedGoogle Scholar
  35. Tanaka S, Kerfeld CA, Sawaya MR et al (2008) Atomic-level models of the bacterial carboxysome shell. Science 319:1083–1086. doi: 10.1126/science.1151458 PubMedCrossRefGoogle Scholar
  36. Tanaka S, Sawaya MR, Phillips M, Yeates TO (2009) Insights from multiple structures of the shell proteins from the beta-carboxysome. Protein Sci 18:108–120. doi: 10.1002/pro.14 PubMedCentralPubMedGoogle Scholar
  37. Wheatley NM, Gidaniyan SD, Liu Y et al (2013) Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins. Protein Sci 22:660–665. doi: 10.1002/pro.2246 PubMedCentralPubMedCrossRefGoogle Scholar
  38. Winn MD, Ballard CC, Cowtan KD et al (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242. doi: 10.1107/S0907444910045749 PubMedCentralPubMedCrossRefGoogle Scholar
  39. Yeates TO, Tsai Y, Tanaka S et al (2007) Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans 35:508–511. doi: 10.1042/BST0350508 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Thomas J. Keeling
    • 1
  • Bożena Samborska
    • 1
  • Ryan W. Demers
    • 1
  • Matthew S. Kimber
    • 1
    Email author
  1. 1.Department of Molecular and Cellular BiologyUniversity of GuelphGuelphCanada

Personalised recommendations