Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Biophysical and structural studies of CtrB, a polysaccharide co-polymerase from Neisseria meningitidis   (#409)

Lorelei Masselot--Joubert 1 , Luke Smithers 1 , Matthew Jackman 2 , Michael J. Landsberg 2 , Alice Vrielink 1
  1. School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia

Polysaccharide co-polymerases (PCP) are a family of proteins involved in the transport of polysaccharides across the inner membrane of Gram-negative bacteria. They are thought to be associated with ABC-transporters however their exact function is not completely understood1. They have 2 transmembrane helices situated in the bacterial inner membrane, as well as a large periplasmic soluble domain. Three subfamilies of PCPs have been defined based on sequence and structural information: PCP-1, PCP-2 and PCP-3. While some structural and functional information is known for PCP-1 and PCP-2 subfamily members, little is known about the PCP-3 subfamily2. Differences in these subfamilies include the extent of homo-oligomerisation and thus structural organisation within the membrane.  The capsule transporter B (CtrB) from Neisseria meningitidis is a PCP-3 subfamily member involved in the transport of capsular polysaccharides through an ABC-transporter dependent system3, 4.

To better understand the structure and function of CtrB, we have carried out biophysical and electron microscopy studies of the recombinant protein in detergent micelles. Size exclusion chromatography (SEC) and multiangle laser light scattering studies show that the sample is homogenous and there are between 3 and 4 molecules in the assembled complex. Transmission electron microscopy of negatively stained particles supports that the protein exists as a trimer in detergent micelles. These results contrast with previous reports suggesting that the protein exists as a pentamer in liposomes4.

For PCP-1 proteins, it has been shown that oligomerisation is environmentally and experimentally-dependant2, therefore, we hypothesized that the native oligomeric assembly of CtrB may require a lipid environment thus we prepared nanodiscs with lipids, assembled CtrB into the discs and assessed the particles by SEC, blue native gel electrophoresis and mass photometry5. Here we present our progress on these studies and provide suggestions for further structural studies of the complex in a more native lipid environment.

  1. L. Cuthbertson, I. L. Mainprize, J. H. Naismith and C. Whitfield, Microbiology and Molecular Biology Reviews 73 (1), 155-177 (2009).
  2. S. Kalynych, M. Cherney, M. Bostina, I. Rouiller and M. Cygler, Protein Science 24 (1), 58-69 (2015).
  3. L. M. Willis and C. Whitfield, Carbohydrate Research 378, 35-44 (2013).
  4. K. Larue, R. C. Ford, L. M. Willis and C. Whitfield, Journal of Biological Chemistry 286 (19), 16658-16668 (2011).
  5. J. Miehling, D. Goricanec and F. Hagn, ChemBioChem Communications 19, 1927-1933 (2018).