An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

Abstract

ABSTRACTA 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 the large 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. 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. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped.Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain —let alone the whole-length protein— has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.