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.
Highlights
sporulation-related repeat (SPOR) domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems
Four proteins with SPOR domains have been identified in Escherichia coli (DamX, DedD, FtsN, and rare lipoprotein A (RlpA)), all of which are involved in cell division, with one (FtsN) assessed as indispensable[12,13]
The gene for the SPOR-RlpA domain of P. aeruginosa was cloned and overexpressed in E. coli and the protein was purified to homogeneity and different synthetic PG fragments (Fig. 2) were produced (Supplementary Table 1, see Materials and Methods)
Summary
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. We document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPORRlpA) by mass spectrometry and structural analyses, and demonstrate that 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. Notwithstanding the availability of NMR structures for the SPOR domains of FtsN21 and DamX from E. coli[20], and of the sporulation protein CwlC of B. subtilis[22], none sheds light on the unique recognition of the denuded glycan, as we will discuss
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