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The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir's binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir's scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion., (© 2024. The Author(s).)

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SO x H-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.

Members of the histone-like nucleoid structuring protein (H-NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H-NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH, or temperature. H-NS oligomerization is essential for its activity. Structural models of different truncated forms are available. However, high-resolution structural details of full-length H-NS and its DNA-bound state have largely remained elusive. We report on progress in characterizing the biologically active H-NS oligomers with solid-state NMR. We compared uniformly ((13)C,(15)N)-labeled ssNMR preparations of the isolated N-terminal region (H-NS 1-47) and full-length H-NS (H-NS 1-137). In both cases, we obtained ssNMR spectra of good quality and characteristic of well-folded proteins. Analysis of the results of 2D and 3D (13)C-(13)C and (15)N-(13)C correlation experiments conducted at high magnetic field led to assignments of residues located in different topological regions of the free full-length H-NS. These findings confirm that the structure of the N-terminal dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition., (© 2013 FEBS.)

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 3-5 units of Ler. DNA wraps around Ler particles that contain more than 15-16 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 degrees C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS.