Together this argued that the majority of mutations leading to YfiBNR activation cluster Bioactive compound in this region of the YfiB protein. Some of these variants had very strong activating effects despite showing severely reduced stability (Figure 4A). Three further, similar substitutions (I40F, V42M, and E45G) were found in conjunction with other singly isolated activating mutations. While neither the V42M nor the E45G substitutions contributed positively to YfiB activity, the YfiB-I40F-F48L allele produced a far stronger phenotype than F48L alone, suggesting that the I40F mutation also contributes to YfiB activation (Figure 4A). When the locations of the activating mutations were plotted onto a 3-D homology model of YfiB, they clustered around the first helix of the PAL domain (Figure 4B).
Interestingly, activating residues in YfiB surround a predicted surface-exposed hydrophobic region, similar to that seen in the model of YfiR (Figure 3C). This hydrophobic patch is highly conserved in YfiB homologs, but absent in the YfiB structural homolog OprL [61], [62]. Most notably, W55, predicted to form the core of the hydrophobic binding site, is very highly conserved, only replaced in a small minority of cases with phenylalanine (note that the YfiB W55L mutation forms a weak SCV in PA01). Figure 4 Mutational analysis of YfiB. To determine whether binding to PG is important for YfiB function, mutants were constructed with two critical peptidoglycan-binding residues [63] replaced with alanine.
Expression of the resulting gene, yfiB-D102A-G105A (PG-) did not induce an SCV colony morphology and had no effect on attachment, despite producing wild type-like levels of protein (Figure 4D, E). To assess the role of the YfiB OM lipid anchor (LA), the YfiB signal peptide was replaced with that from YfiR, which lacks the Cys lipid acceptor residue. The resulting mutant (LA-) slightly increased attachment upon induction in an YfiN-dependent manner (Figure 4D). This residual activity was dependent on peptidoglycan binding, as a PG-/LA- mutant was fully inactive (Figure 4D). These data suggest that peptidoglycan binding and, to a lesser extent, membrane anchoring are required for YfiB activity. When the mutations disrupting PG binding were combined with activating mutations in yfiB (F48S or L43P; see above), very low YfiB protein levels were detected, possibly as a result of reduced protein stability (Figure 4D).
Importantly, the resulting alleles still led to increased attachment and generated an SCV phenotype (Figure 4D, E). This suggested that the activating mutations are dominant over the loss of peptidoglycan binding, and that they are able to fix YfiB in its active conformation independent of PG binding. YfiB sequesters Cilengitide YfiR at the outer membrane (Figure 1). To examine the relationship between this activity and YfiB function, membrane fractionation was carried out for strains expressing PG- and hyperactive (F48S) YfiB variants alongside an yfiR-flag allele.
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