The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

Abstract

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.

Molecular insights into phosphoethanolamine cellulose formation and secretion

“…6. Our putative BcsC-cellotetraose complex indicates cellulose translocation along the solenoid, consistent with recent insights into poly N-acetylglucosamine interactions with the TPR of PgaA 41 . Cellulose migrating away from the solenoid helix would likely be irreversibly mislocalized, thereby stalling cellulose biosynthesis.…”

The deficiency of poly-β-1,6-N-acetyl-glucosamine deacetylase trigger A. baumannii to convert to biofilm-independent colistin-tolerant cells

“…One is the smaller TPR domain in BbPgaA than EcPgaA, which possess the ability to interact with PgaB to affect its PNAG translocation activity. It was confirmed by further study of protein-protein interaction between the TPR domain of PgaA and PgaB 30 . The other possibility is the deficient residues in the BbPgaA TPR domain may lead to the porin structure constitutive open for PNAG translocation 38 .…”

“…3). High acetyl level of PNAG appears to bind to PgaA with a less extent against exopolysaccharide export 30 , reflecting the less biofilm production in ΔAbpgaB1ΔAbpgaB2 (Fig. S5).…”

Structural and functional analysis ofPseudomonas aeruginosaPelA provides insight into the modification of the Pel exopolysaccharide

“…As AF2 is unable to predict conformational changes, the current AF2 model only enables a mechanism for the deacetylation of Pel to be predicted. In E. coli, the dual-active protein PgaB first partially deacetylates the PNAG polymer (26).…”

“…Interactions between modification enzymes and the periplasmic TPRs of outer membrane porin proteins are a common feature of synthase-dependent exopolysaccharide biosynthetic pathways. For example, the 4 modification and export of poly-β(1,6)-N-acetylglucosamine (PNAG) in E. coli and alginate in P. aeruginosa is coupled through the PgaB-PgaA and AlgX-AlgK-AlgE interactions, respectively (22)(23)(24)(25)(26).…”