Residues interacting with moenomycin and structure-based sequence alignment of transglycosylases. (A) The potential hydrogen-bonding interactions (distance cutoff 3.2 Å) between E. coli PBP1b and moenomycin are shown as dashed lines in black. The interactions between the putative active sites (E233 and E290) and moenomycin proposed in ref. 6 are shown as dashed lines in red. (B) Comparison of moenomycin-binding modes between E. coli PBP1b and SaPBP2 (Left); between E. coli PBP1b and AaPGT (Right). TG and residues of E. coli PBP1b, SaPBP2, and AaPGT are shown in red, cyan, and green, respectively. Moenomycin are shown in light gray (for E. coli PBP1b) and dark gray (for SaPBP2 and AaPGT). (C) Sequences of TG from E. coli PBP1b, SaPBP2, and AaPGT are aligned according to their secondary structure elements. Residues forming the potential interaction with moenomycin are shaded in red.

UB2H domain, its deletion phenotype and pull-down assay. (A) The structurally homologous domains from PBP1b, UvrB (PDB ID code 2NMV), and TRCF (PDB ID code 2EYQ) are shown in yellow, cyan, and magenta, respectively. The nonhomologous parts of these proteins are colored in gray. (B) Morphological differences and DNA segregation between the wild type and UB2H-truncated (PBP1bΔUB2H) strains are shown in differential interference contrast microscopy combined with DAPI staining images. (Scale bar, 1 μm.) (C) Wild-type PBP1b, deletion mutant PBP1bΔUB2H, and UB2H domain only were coupled onto the CNBr-activated Sepharose, and their binding ability to MltA, PBP3, and FtsN was examined.

Overall structure and topology of E. coli PBP1b. (A) The crystal structure of PBP1b is represented as a ribbon diagram. The TM, UB2H, TG, and TP domains are color coded in cyan, yellow, red, and blue, respectively. Moenomycin is represented as van der Waals spheres. Tryptophan and tyrosine residues located near the water–membrane interfaces are shown in black sticks. The proposed membrane location is indicated by a gray rectangle. All figures of 3D structural representations were made with PyMOL (www.pymol.org). (B) The 1D and 2D topology of E. coli PBP1b are color-coded as in A. The numbering (1–5) at the N terminus of UB2H domain and the alphabet (A–E) at the C terminus of UB2H domain are markers for the locations used in the combinatorial domain deletion strategy (see SI Materials and Methods).

Interdomain flexibility and a model of peptidoglycan synthesis. (A) E. coli PBP1b and 2 SaPBP2 conformers (6, 23) were indicated by colored, light gray (PDB ID code 3DWK), and dark gray (PDB ID code 2OLV), respectively. The TG domain of SaPBP2 was superimposed onto the TG of E. coli PBP1b. Side view (Left) and top view (Right) of the comparison reveals possible flexibility of a hinge region between TG and TP domains. (B) Active sites of TG and TP were shown by van der Waals spheres. The disaccharide, pentapeptide, and lipid tail of lipid II and peptidoglycan were shown in orange surface, green surface, and blue line, respectively. A single strand of the proposed peptidoglycan model (25) was docked onto the structure of E. coli PBP1b, with lipid IV portion replacing moenomycin. The incoming lipid II, of which the chemical structure is shown on top, diffuses in the plane of the membrane. After the TG reaction, the lipid moiety of this lipid II is kept as the membrane anchor, whereas the original lipid tail (shown as dotted line) is recycled. The polymerized peptidoglycan grows perpendicularly to the membrane and toward the TP domain, where the cross-linking reaction of the pentapeptides takes place (see also Movie S3).