Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein

Abstract

The opportunistic pathogen P. aeruginosa utilizes a type III secretion system (T3SS) to support acute infections in predisposed individuals. In this bacterium, expression of all T3SS-related genes is dependent on the AraC-type transcriptional activator ExsA. Before host contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The repression, thought to occur through direct interactions between the two proteins, is relieved upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC sequesters ExsD. We have solved the crystal structure of Delta20ExsD, a protease-resistant fragment of ExsD that lacks only the 20 amino terminal residues of the wild-type protein at 2.6 A. Surprisingly the structure revealed similarities between ExsD and the DNA binding domain of transcriptional repressor KorB. A model of an ExsD-DNA complex constructed on the basis of this homology produced a realistic complex that is supported by the prevalence of conserved residues in the putative DNA binding site and the results of differential scanning fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts through interactions with ExsA and raise new questions with respect to the underlying mechanism of ExsA regulation.

Figure 1

(a) Two orthogonal views of the rainbow-colored model of the Δ20ExsD crystal structure. Secondary structure elements are labeled and the termini are marked. (b) Reflecting the reported solution state of ExsD there are three molecules in the asymmetric unit. Protein–protein contact surfaces within the trimer amount to about 2000 Ų for each molecule and involve all three sections of the protein. This figure was generated by PYMOL.

Figure 2

(a) Superposition of Δ20ExsD with the KorB-DNA complex. Two subdomains of transcriptional repressor KorB interact with the major grove of the operator sequence. The amino terminal domain of Δ20ExsD encompassing residues 37–108 and helices α1–α5 is structurally homologous to the carboxy-terminal domain of KorB (amino acids 194–252), which has been shown to confer specificity to the KorB-DNA interactions. DALI reported a Z-score of 5.2 and an RMSD of 2.6 Å for the backbone atoms of the 52 overlapping residues. (b) Electrostatic surface presentation of Δ20ExsD. The DNA molecule of the KorB-complex is also shown to mark the putative DNA binding site on Δ20ExsD. ExsD has a calculated isoelectric point of 5.15 and consequently displays large areas with negative surface potential. Remarkably, the putative DNA binding site of Δ20ExsD coincides with the only area of the protein with a significant concentration of positively charged residues. The arginine residues discussed in the text, such as R51, R58, and R86, are highlighted in the figure. (c) Close-up view of the second putative DNA binding site of ExsD. The cyan-colored section encompassing residues 112–120 appears poised for an interaction with the DNA backbone. Also highlighted are amino acids 217–226. This region would be positioned directly above the major groove of the DNA and might also be involved in binding. It is noteworthy that, although the surrounding areas are rich in acidic residues, no negatively charged residues are contained within either of the highlighted regions.

Figure 3

Sequence alignment of known ExsD orthologs. The secondary structure elements obtained from the Δ20ExsD structure are displayed above the sequence. Residues that constitute part of the ExsD-DNA interface in the modeled complex are marked by a triangle below the sequence.

Figure 4

The ExsD trimer is incompatible with DNA binding. (a) Structure of the Δ20ExsD trimer with a single DNA molecule placed into the DNA binding site of molecule C (yellow). No space is available for the binding of two additional DNA molecules and the modeled-in DNA clashes with the two other ExsD molecules. (b) Results of DSF studies for (full length) ExsD-dsDNA interactions. For 10 μM ExsD alone a T_m of 49.7°C ± 0.3°C was observed, while 10 μM ExsD combined with 100 μM of 24-nucleotide double-stranded DNA produced a T_m of 37.7°C ± 0.2°C. The observed decrease in T_m is consistent with the predicted dissociation of the ExsD trimer upon DNA binding.