Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Pore structure controls stability and molecular flux in engineered cages (#173)

Lachlan S.R. Adamson 1 2 , Nuren Tasneem 1 , Michael P Andreas 3 4 , William Close 5 , Tobias W Giessen 3 4 , Yu Heng Lau 1
  1. School of Chemistry, University of Sydney, Sydney, NSW, Australia
  2. CSIRO Future Science Platform in Synthetic Biology, Commonwealth Scientific and Industrial Research Organisation, Brisbane, QLD, Australia
  3. Department of Biomedical Engineering, University of Michigan Medical School, Ann Arbor, MI, USA
  4. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, Australia
  5. Center for Microscopy & Microanalysis, University of Queensland, Brisbane, QLD, Australia

Encapsulins are a recently discovered class of protein cages that are used widely by prokaryotes as organelles. Encapsulin systems consist of a porous protein cage that is formed from multiple copies of a single shell protein, and a cargo protein that possesses a targeting peptide that localises the cargo within the cage.1 There have been numerous reported efforts to engineer encapsulins to form nanoreactors and synthetic organelles, owing to the simplicity of encapsulins and their ability to be expressed heterologously.2 The application of encapsulin nanoreactors is limited by the selectivity for diffusing chemicals that the native cage possesses. Engineering encapsulin cages to alter the diffusion selectivity could enable more sophisticated functions in nanoreactors and synthetic organelles. A detailed understanding of the principles that govern the diffusion of chemicals through encapsulin cage pores is required to alter their diffusion selectivity. Previous work reported that the pores of the Thermotoga maritima encapsulin were amenable to mutations, and that deletions in the pore-forming loop region resulted in wider pores and an increase in ion flux.3 However, there has been no investigation into the effects of altered pore charge on ion flux and selectivity.

In this work, 24 T. maritima encapsulin mutants with pores of varying size and charge were designed. Twelve of the mutants were able to be purified and characterised for assembly and thermal stability. Cryo-EM structures of seven of the successful mutants were obtained, and stopped-flow and molecular dynamics experiments were used to analyse effects of the pore mutations on ion flux through the cages.

References:

  1. Giessen, T. W.; Silver, P. A., Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol 2017, 2 (6), 1-11.
  2. Giessen, T. W., Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science. Curr. Opin. Chem. Biol. 2016, 34, 1-10.
  3. Williams, E. M.; Jung, S. M.;  Coffman, J. L.; Lutz, S., Pore Engineering for Enhanced Mass Transport in Encapsulin Nanocompartments. Acs Synth Biol 2018, 7 (11), 2514-2517.