Structural Insights into Bacteriophage GIL01 gp7 Inhibition of Host LexA Repressor

Abstract

Highlights d Crystal structure of GIL01 gp7 has been solved d A hybrid approach provides a model for gp7 scaffolding of LexA d gp7 is seen to interact with phylogenetically distinct Staphylococcus aureus LexA d Structural evidence of a phage factor associating with LexA to modulate the SOS response

Homodimerization and heterodimerization requirements of Acinetobacter baumannii SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses

“…If pre-existing DdrR dimers are important in forming an association with UmuDAb, our BACTH system conditions, which contained only one form of a DdrR-AC fusion protein (i.e., with either the N-or C-terminus free, but not a combination of both) to interact with UmuDAb, would have prevented observing a UmuDAb-DdrR interaction. Furthermore, this possibility is consistent with the crystal structure of the scaffold protein gp7 forming similar dimers before its association with LexA, where the N terminus of one monomer associates with the C terminus of the other monomer in a dimeric fourhelix bundle facilitated by interactions between beta-strands of each gp7 monomer (Caveney et al 2019). As two similarly sized and organized helices and beta-strands were also predicted with TM-align modeling (Zhang and Skolnick 2005) to exist in a similar configuration in the Cterminus of DdrR (Fig.…”

“…The observations of the greater sensitivity seen in the colorimetric assay is consistent with previous work by Battesti and Bouveret, 2012. UmuDAb represses SOS genes like LexA (Norton et al 2013;Aranda et al 2013;Hare et al 2014). Based on interactions between the small protein gp7and LexA that facilitate LexA-DNA binding (Caveney et al, 2019), we hypothesized that the 9 kD DdrR interacts with UmuDAb in a similar manner. We tested all eight possible fusion protein configuration pairings for interaction between hybrid UmuDAb-AC and DdrR-AC proteins ( Table 2).…”

“…2b). This sized structure is similar to the N-terminal 36 amino acids of gp7 (7 kD), which contains two 16-and 12-amino acid alpha helices separated by a three amino acid beta strand (shown in the crystal structure 6N7O in the RCSB Protein Data Bank (Caveney et al 2019)). Although these I-TASSER models both showed a similar structure, none of the PDB hits of threading templates were shared for these two proteins, i.e., DdrR was not directly modeled on gp7.…”

“…We tested whether a hybrid DdrR-AC interacted with another DdrR-AC protein, possibly in a manner reminiscent of how RecA forms filaments (Stasiak and Egelman 1986) or how the bacteriophage GIL01 gp7 protein forms dimeric four-helix bundles to mediate LexA-DNA interactions in Bacillus thuringiensis (Caveney et al 2019). The four possible pairs of DdrR hybrid proteins were tested for DdrR self-interaction.…”

“…One One precedent for repressor action being affected by a small coregulatory protein exists in the LexA repressor and the gp7 bacteriophage protein pair. Gp7, like DdrR, is a small (6 kD) protein, which regulates the lysogenic cycle of the temperate bacteriophage GIL01 that encodes it, via physical interactions of gp7 dimers with the chromosomally encoded LexA repressor (Fornelos et al 2015;Caveney et al 2019). This example suggests the hypothesis that UmuDAb and DdrR coregulate gene expression through physical interaction, which we sought to test in this study.…”