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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. Author summary: Exopolysaccharides are an important component of the extracellular matrix of bacterial and fungal biofilms and provide protection against the host immune response and antibiotics. In Gram-negative bacteria, these polymers are synthesized in the inner membrane and translocated across the periplasm before being secreted across the outer membrane. The periplasm presents both a challenge as an additional environment to cross and an opportunity to chemically alter the polymer prior to secretion to render it more effective. This study focuses on a periplasmic alpha-helical repeat domain whose wide-spread homologues are involved in the export of many chemically distinct exopolysaccharides. We found that in E. coli this superhelical TPR domain acts as a scaffold that can bind the polymer PNAG and alter the enzymatic activity of PgaB, thus providing a means to affect the deacetylation level and chain length of the secreted polymer. Scaffold proteins are known as binding hubs within cellular pathways that often have a central regulatory function and facilitate evolution due to their repetitive modular building blocks. Our study sheds light on the principles of polysaccharide modification and export, which we hope will promote the development of applications against bacterial infections. [ABSTRACT FROM AUTHOR]