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

Abstract

A revised model of the aromatic binding A domain of the σ 54 -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.

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida

“…To make structural sense of the diverse genetic and phenotypic data above, the 3D model of the XylR A domain prepared previously by the I‐TASSER server (Dvořák et al, 2021 ) was further refined using the recently available AlphaFold structure prediction software (Senior et al, 2020 ). While the earlier and updated models differed only minimally (Figure S2 ), the predicted AlphaFold structure enabled an easy calculation and spatial location of the amino acid changes borne by the variants studied above.…”

“…Changes in this region typically originate a semi‐constitutive phenotype (Garmendia & de Lorenzo, 2000 ) and they are believed to reflect alterations in the transmission of conformational changes to the rest of the protein after inducer binding to the A domain. V101 has an additional substitution in the highly conserved Y159 (Dvořák et al, 2021 ; Galvão et al, 2007 ). The Y159F variant lacks a hydrogen bond which probably stabilizes helices forming the bottom of the binding pocket and to which Y159 contributes (Dvořák et al, 2021 ).…”

“…V101 has an additional substitution in the highly conserved Y159 (Dvořák et al, 2021 ; Galvão et al, 2007 ). The Y159F variant lacks a hydrogen bond which probably stabilizes helices forming the bottom of the binding pocket and to which Y159 contributes (Dvořák et al, 2021 ). Y159F may thus cause the loosening of the binding pocket (Dvořák et al, 2021 ) and the combination of the two mutations in XylR V101 results in the high induction phenotype observed here and in the previous study of (Galvão et al, 2007 ).…”

“…The Y159F variant lacks a hydrogen bond which probably stabilizes helices forming the bottom of the binding pocket and to which Y159 contributes (Dvořák et al, 2021 ). Y159F may thus cause the loosening of the binding pocket (Dvořák et al, 2021 ) and the combination of the two mutations in XylR V101 results in the high induction phenotype observed here and in the previous study of (Galvão et al, 2007 ). The rest of the substitutions (M14I, S47R, L110S, I129N) do not appear in residues conserved in the family of XylR‐like transcriptional regulators (Devos et al, 2002 ).…”

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria

“…Furthermore, σ 54 also regulates the susceptibility to tobramycin, quinolones, and carbapenems [ 11 , 12 , 13 , 14 ]. In P. putida , it also affects the utilization of nitrate, urea, and uncharged amino acids as nitrogen sources, as well as of lysine, C 4 -dicarboxylates, and alpha-ketoglutarate as carbon sources [ 15 , 16 ]. In addition, σ 54 factor controls bacterial growth [ 17 , 18 , 19 ], nitrogen and carbohydrate assimilation [ 20 , 21 ], swimming and twitching motility [ 22 ], biofilm formation [ 23 , 24 ], exopolysaccharide (EPS) production [ 23 ], quorum sensing [ 25 , 26 ], type VI secretion systems (T6SS) [ 27 , 28 ], virulence [ 29 , 30 ], environmental adaptation [ 31 , 32 ], and antibiotic resistance [ 33 , 34 ] in other bacteria.…”