Abstract
Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.