Strategies for the engineering of allosteric proteins, which are proteins that bind ligands at a specific site other than the reaction site and affect the reaction activity, are still being perfected. There have been allosteric proteins successfully engineered based on the hypothesis that the two allosterically related sites are distinct, modular domains and use trial and error to construct and test novel protein domain fusions for allostery. This work uses laboratory evolution to engineer the peptide binding affinity of the protein binding domain of the allosteric E. coli protease DegS. The protein binding domain is a PDZ domain (named for Postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and Zonula occludens-1 (ZO-1)) that binds the C-terminus of unfolded outer membrane porins. Combinatorial libraries of PDZ domain variants were displayed anchored to the periplasmic membrane of E. coli. The cells were permeabilized and incubated with fluorescent peptide ligands. PDZ domains were screened by flow cytometry for binding to the target peptide ligands. The PDZ domain binding affinity was improved by 20-fold for the peptide ligand that represents the physiological ligand; and the PDZ domain binding affinity was expanded to accommodate a negatively charged residue in a novel peptide ligand. The E. coli anchored peripalsmic expression (APEx) methodology in conjunction with flow cytometry had not previously been used to modify the binding affinity of a PDZ domain. The selected PDZ domain variants were then fused to the wild-type DegS protease domain and analyzed to determine if allosteric activation was made more sensitive to the native ligand or altered to respond to the novel peptide ligand. Interestingly, the DegS fusion protein with the PDZ variant containing the most subtle mutations retained a degree of allostery for the physiological peptide ligand and obtained a degree of allostery for the novel activating peptide ligand. Other selected PDZ variants with additional and expected mutations in the ligand binding site did not respond allosterically to the peptide ligands and the respective DegS fusions were constitutively active, suggesting that the amino acid network linking the allosteric binding event to protease activity is intricately integrated. / text

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