Figure 7. PBP1 inhibits the synergistic hydrolysis of cell wall by RipA-RpfB complex.

N-terminal GST fusion proteins were expressed and purified from E. coli. Equal amounts of individual or combinations of proteins were incubated with insoluble FITC-labeled substrate: M. luteus cell wall material (A) or Streptomyces peptidoglycan (B). The extent of hydrolysis was determined by measuring the amount of soluble FITC remaining after centrifugation, and thus released during hydrolysis of the insoluble substrate. GST and buffer alone were used to determine background release of FITC and were subtracted from final values. Data are from representative experiments, each done in triplicate. Data are represented as mean +/− SEM. p-values for one-tailed, unpaired t-tests: (A) 1 vs. 2: 0.027 significant, 2 vs. 4: 0.016 significant, 1 vs. 3: 0.074 not significant, 1 vs. 4: 0.188 not significant, (B): 1 vs. 2: 0.009 significant, 2 vs. 4: 0.018 significant, 1 vs. 3: 0.279 not significant, 1 vs. 4: 0.240 not significant (significant to p<0.05). (C) Schematic diagram of the basic structure of mycobacterial peptidoglycan, indicating where RpfB and RipA are predicted to have hydrolytic activity and where PBP1 is predicted to have synthetic activity. Lines connecting NAG to NGM represent β-1,4-glycosidic bonds, while lines connecting NGM to NGM represent peptide cross-linkages. NAG: N-acetyl glucosamine, NGM: N-glycolyl muramic acid, NAM: N-acetyl muramic acid (note that mycobacteria have a mixture of NGM and NAM, with the NGM structure shown here).