Structural glycobiology and inhibition of S-layer anchoring

2D-crystalline protein lattices are cell surface structures (surface (S-) layers) of many bacteria and are potential targets for therapeutic inhibition. Many S-layers are modified with carbohydrate chains (glycosylation) and can be attached to the cell wall through the interaction of a terminal S-layer homology (SLH) domain trimer with a secondary cell wall glycopolymer (SCWP). Current insight in the SLH domain trimer-SCWP interactions stems from recombinant, non-glycosylated protein and short synthetic SCWP fragments. As demonstrated for the model organism Paenibacillus alvei, a terminal pyruvylated N-acetylmannosamine residue of SCWP is essential for cell wall binding and two active binding grooves are provided by the SLH domain trimer in a mutually exclusive manner. We hypothesize that the native glycosylation of the P. alvei SLH domain trimer located in the two binding grooves influences the molecular logic of S-layer anchoring to the cell wall and that the demonstrated terminal pyruvylated N-acetylmannosamine S-layer binding epitope for SCWP is an ideal starting point to design inhibitors of proper cell wall assembly. We will analyze the influence of the glycosylation of the SLH domain trimer on binding to SCWP in a bottom-up approach of increasing complexity and aim to identify small molecule inhibitors of the SLH domain trimer-SCWP interaction. Methods include chemical synthesis of SCWP fragments and tailored analogues, rational S-layer (glyco)protein engineering, P. alvei cell design, biophysical protein-carbohydrate interaction analyses, X-ray crystallography; cryo-electron tomography/microscopy, and molecular modelling and simulation. This interdisciplinary project will yield a detailed mechanistic model of S-layer anchoring in Gram-positive bacteria. A molecular understanding of the dynamic cell wall anchoring mechanism of S-layer glycoproteins is key to exploiting the therapeutic or biotechnological potential of these frequent bacterial cell surface structures. This project is based on complementary expertise of Christina Schäffer (PI; molecular (glyco)microbiology, biochemistry), Chris Oostenbrink (molecular modelling, molecular dynamics simulation), Markus Blaukopf (chemical synthesis, NMR)-all Universität für Bodenkultur Wien, AUT-Stephen V. Evans (X-ray crystallography), University of Victoria, CAN, and Tanmay A. Bharat (cryo-electron tomography, cryo-electron microscopy), University of Cambridge, UK.