Structural basis of denuded glycan recognition by SPOR domains in bacterial cell division

Abstract

SPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.

SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

“…The SPOR domain is needed to efficiently target DamX to the division site and to cause cell division inhibition when DamX is overproduced (40). Together, these results suggest that DedD and DamX might recognize and bind to specific structures in PG present at the division site before the septum synthesis starts, which could be either denuded glycan strands (28) or perhaps the 1,6-anhydro ends of glycan strands (33,36). Later, during septation, SPOR proteins provide a connection between PG synthases and the inward-growing septal PG, which may function to stabilize the constricting cell envelope and/or regulate PG synthesis.…”

“…SPOR domain protein structures have been determined for two of the four proteins in E. coli : FtsN (PDB ID 1UTA ) ( 39 ) and DamX (PDB ID 2LFV ) ( 40 ). Additionally, structures of the P. aeruginosa homologue of RlpA, both in the apo form and in complex with denuded glycans (PDB IDs 6I05 , 6I09 , 6I0N , and 6I0A ) ( 28 ), and the sporulation-specific CwlC from Bacillus subtilis (PDB ID 1X60 ) ( 41 ) have been determined. In the context of SPOR proteins that exist in E. coli , only DedD has not been structurally characterized.…”

“…(B) Structured regions are depicted as a ribbon diagram with secondary structure elements shown, colored in rainbow from the N terminus (blue) to the C terminus (red). The SPOR domain regions of (i) E. coli FtsN (yellow; PDB.ID 1UTA ) ( 39 ), (ii) E. coli DamX (gray; PDB.ID 2LFV ) ( 40 ), (iii) P. aeruginosa RlpA (red; PDB.IDs 6I05 ) ( 28 ), and (iv) B. subtilis CwlC (green; PDB.ID 1X60 ) ( 41 ) aligned to the structured SPOR domain region of E. coli DedD (C). In each case, DedD is in purple.…”

“…This level of atomistic variability is mostly observed in the pair of α-helices that act to scaffold the β-sheet, while the β-sheet itself is more structurally conserved. This is perhaps unsurprising, as the β-sheet region was seen to form the majority of the binding interface in the liganded structures of P. aeruginosa RlpA ( 28 ). To further unravel the potential similarities and differences in the binding mode of DedD in comparison to other SPOR proteins, we generated a model of liganded DedD.…”

“…These SPOR proteins bind peptidoglycan and are widely conserved among bacteria ( 27 ). Recent structural work explained their ability to bind denuded glycan strands ( 28 ). In E. coli , the SPOR proteins localize to division septa when amidases are present and show a stronger septal localization signal in mutants lacking lytic transglycosylases, supporting their binding to denuded glycan strands ( 29 ).…”