Structure-Guided Investigation of Lipopolysaccharide O-Antigen Chain Length Regulators Reveals Regions Critical for Modal Length Control

Abstract

We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.

Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation

“…3 and Table 2), suggesting that maintenance of the negative charge at this position is critical. This correlates with data by Kalynych et al (2011), whereby substitution or deletion of the FepE amino acid region 256-273 (where D268 is located) resulted in shortened LPS Oag chain lengths. We report for the first time two specific residues, L168 and D268, which are essential for FepE function in LPS Oag modal chain length determination.…”

“…Notably, two of these positions, D268 and G274, are located in the FepE region spanning amino acid residues 240-299 which correlates to the WzzB SF segment spanning residues 200-255 based on sequence alignment (Tocilj et al, 2008). Kalynych et al (2011) showed that this functionally important WzzB SF segment was predominantly located on the external surface of the WzzB SF oligomer and hypothesized that the external surface of chain length regulators might represent the principal site of modal length control. Residues D268 and G274 located within this same region of FepE, however, point towards the internal cavity of the oligomer and still affect LPS Oag modal chain length (Figs 2 and 3).…”

“…1), both proteins are structurally similar (Kalynych et al, 2011(Kalynych et al, , 2012Tocilj et al, 2008) and belong to the same family of PCP1a proteins. Analysis of several WzzB SF mutants showed that mutational alterations made between amino acids 102-107, 128-131 and 219-232 consistently had an effect on Oag modal chain length determination (Daniels & Morona, 1999;Papadopoulos & Morona, 2010;R.…”

“…As PCP proteins are predicted to share similar structural folds (Tocilj et al, 2008), these studies suggest that specific amino acids rather than specific regions of PCP1a proteins affect regulation of LPS Oag modal chain length. More recently, a study using in-frame linker insertion mutagenesis on WzzB SF identified periplasmic sites which altered the LPS Oag modal chain length to varying degrees (Papadopoulos & Morona, 2010) (Kalynych et al, 2011). To date, no additional sitedirected mutagenesis has been undertaken to identify specific amino acids that affect FepE regulation of LPS Oag modal chain length.…”

“…Several studies on FepE and other PCP1a proteins have been undertaken to define functional regions that affect LPS Oag modal chain length determination (Daniels & Morona, 1999;Franco et al, 1998;Kalynych et al, 2011;Kintz & Goldberg, 2011;Marolda et al, 2008;Papadopoulos & Morona, 2010;Purins et al, 2008;Tocilj et al, 2008). Franco et al (1998) showed that site-directed mutations made in the periplasmic region of E. coli O2 Wzz at residues D90 and L91 altered LPS Oag modal chain length.…”