À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors

To investigate the activation mechanism of the enhancer-binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled N-terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure. When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g. nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors. Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulators à la carte that are responsive to predetermined, natural or xenobiotic chemical species.     

An updated structural model of the A domain of the Pseudomonas putida XylR regulator poses an atypical interplay with aromatic effectors

A revised model of the aromatic binding A domain of the σ⁵⁴-dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR and DmpR. The resulting frame was instrumental for mapping a number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. Yet, several loop regions of the A domain could provide the flexibility required for widening such a tunnel for passage of aromatic ligands. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Structural and proteomic analyses confirmed the predicted in/out distribution of residues but also supported two additional possible scenarios of interaction of the A domain with aromatic effectors: a dynamic interaction of the fully structured yet flexible protein with the aromatic partner and/or inducer-assisted folding of the A domain.     

Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250.

Degradation of phenol by Acinetobacter calcoaceticus NCIB8250 involves (sigma54-dependent expression of a multicomponent phenol hydroxylase and catechol 1,2-dioxygenase encoded by the mop operon. Complementation of a new mutant deficient in phenol utilization yielded the regulatory locus mopR. It is located in divergent orientation next to the mop operon. MopR is constitutively expressed at a low level from a sigma70-type promoter and belongs to the NtrC family of regulators. The amino acid sequence is similar to that of XylR regulating xylene degradation and to that of DmpR regulating dimethylphenol degradation in Pseudomonas spp. However, it shows a different effector profile for substituted phenols than DmpR. MopR activates phenol hydroxylase expression in the presence of phenol in Escherichia coli, indicating that it binds the effector. The phenol binding A domains of MopR and DmpR have fewer identical residues than the A domains of DmpR and XylR, despite the fact that XylR recognizes different effectors. This suggests that sequence conservation in the A domain does not reflect the potential to bind the respective effectors. Overexpression of the MopR A domain in the presence of wild-type MopR causes loss of mop inducibility by phenol, establishing its negative transdominance over MopR. Deletion of 110 residues from the N terminus did not affect transdominance of the truncated domain, whereas deletion of 150 residues abolished it completely. This result establishes the distinction of two subdomains, A(N) and A(C), which together constitute the A domain. The C-terminal portion of the A domain, A(C), shows considerable affinity for the C domain, even in the presence of the trigger phenol.