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The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de- N- acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Poly-glucosamine subunit B (PgaB) is responsible for the de—N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de—N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB 310—672 ) directly binds PNAG oligomers with dissociation constants of ∼1—3 mM, and the structures of PgaB 310—672 in complex with β-1,6-(GlcNAc) 6 , GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de—N-acetylated PNAG (dPNAG). Furthermore, PgaB 310—672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB 42—655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB 310—672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.

Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (...) directly binds PNAG oligomers with dissociation constants of ~1-3 mM, and the structures of ... in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, ... contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous ... structures and is highly dynamic in the MD simulations. We propose that conformational changes in ... mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release. (ProQuest: ... denotes formulae/symbols omitted.)