Structural Insights into Regulation of Vibrio Virulence Gene Networks.

One of the best studied aspects of pathogenic Vibrios are the virulence cascades that lead to the production of virulence factors and, ultimately, clinical outcomes. In this chapter, we will examine the regulation of Vibrio virulence gene networks from a structural and biochemical perspective. We will discuss the recent research into the numerous proteins that contribute to regulating virulence in Vibrio spp such as quorum sensing regulator HapR, the transcription factors AphA and AphB, or the virulence regulators ToxR and ToxT. We highlight how insights gained from these studies are already illuminating the basic molecular mechanisms by which the virulence cascade of pathogenic Vibrios unfold and contend that understanding how protein interactions contribute to the host-pathogen communications will enable the development of new antivirulence compounds that can effectively target these pathogens.

Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS.

ToxR is a transmembrane transcription factor that is essential for virulence gene expression and human colonization by Vibrio cholerae. ToxR requires its operon partner ToxS, a periplasmic integral membrane protein, for full activity. These two proteins are thought to interact through their respective periplasmic domains, ToxRp and ToxSp. In addition, ToxR is thought to be responsive to various environmental cues, such as bile salts and alkaline pH, but how these factors influence ToxR is not yet understood. Using NMR and reciprocal pull down assays, we present the first direct evidence that ToxR and ToxS physically interact. Furthermore, using NMR and DSF, it was shown that the bile salts cholate and chenodeoxycholate interact with purified ToxRp and destabilize it. Surprisingly, bile salt destabilization of ToxRp enhanced the interaction between ToxRp and ToxSp. In contrast, alkaline pH, which is one of the factors that leads to ToxR proteolysis, decreased the interaction between ToxRp and ToxSp. Taken together, these data suggest a model whereby bile salts or other detergents destabilize ToxR, increasing its interaction with ToxS to promote full ToxR activity. Subsequently, as V. cholerae alkalinizes its environment in late stationary phase, the interaction between the two proteins decreases, allowing ToxR proteolysis to proceed.

Reconstitution of homomeric GluA2(flop) receptors in supported lipid membranes: functional and structural properties.

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

A Stochastic Kinematic Model of Class Averaging in Single-Particle Electron Microscopy.

Single-particle electron microscopy is an experimental technique that is used to determine the 3D structure of biological macromolecules and the complexes that they form. In general, image processing techniques and reconstruction algorithms are applied to micrographs, which are two-dimensional (2D) images taken by electron microscopes. Each of these planar images can be thought of as a projection of the macromolecular structure of interest from an a priori unknown direction. A class is defined as a collection of projection images with a high degree of similarity, presumably resulting from taking projections along similar directions. In practice, micrographs are very noisy and those in each class are aligned and averaged in order to reduce the background noise. Errors in the alignment process are inevitable due to noise in the electron micrographs. This error results in blurry averaged images. In this paper, we investigate how blurring parameters are related to the properties of the background noise in the case when the alignment is achieved by matching the mass centers and the principal axes of the experimental images. We observe that the background noise in micrographs can be treated as Gaussian. Using the mean and variance of the background Gaussian noise, we derive equations for the mean and variance of translational and rotational misalignments in the class averaging process. This defines a Gaussian probability density on the Euclidean motion group of the plane. Our formulation is validated by convolving the derived blurring function representing the stochasticity of the image alignments with the underlying noiseless projection and comparing with the original blurry image.

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons.

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC's. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

An Enantiodefined Conformationally Constrained Fatty Acid Mimetic and Potent Inhibitor of ToxT.

The chiral conformation that palmitoleic acid takes when it is bound to ToxT, the master regulator of virulence genes in the bacterial pathogen Vibrio cholerae, was used as inspiration to design a novel class of fatty acid mimetics. The best mimetic, based on a chiral hydrindane, was found to be a potent inhibitor of this target. The synthetic chemistry that enabled these studies was based on the sequential use of a stereoselective annulative cross-coupling reaction and dissolving metal reduction to establish the C13 and C9 stereocenters, respectively.

Publisher Correction: A disulfide constrains the ToxR periplasmic domain structure, altering its interactions with ToxS and bile-salts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A disulfide constrains the ToxR periplasmic domain structure, altering its interactions with ToxS and bile-salts.

ToxR is a transmembrane transcription factor that, together with its integral membrane periplasmic binding partner ToxS, is conserved across the Vibrionaceae family. In some pathogenic Vibrios, including V. parahaemolyticus and V. cholerae, ToxR is required for bile resistance and virulence, and ToxR is fully activated and protected from degradation by ToxS. ToxS achieves this in part by ensuring formation of an intra-chain disulfide bond in the C-terminal periplasmic domain of ToxR (dbToxRp). In this study, biochemical analysis showed dbToxRp to have a higher affinity for the ToxS periplasmic domain than the non-disulfide bonded conformation. Analysis of our dbToxRp crystal structure showed this is due to disulfide bond stabilization. Furthermore, dbToxRp is structurally homologous to the V. parahaemolyticus VtrA periplasmic domain. These results highlight the critical structural role of disulfide bond in ToxR and along with VtrA define a domain fold involved in environmental sensing conserved across the Vibrionaceae family.

Breaking the bottleneck: eukaryotic membrane protein expression for high-resolution structural studies.

The recombinant expression of eukaryotic membrane proteins has been a major stumbling block in efforts to determine their structures. In the last two years, however, five such proteins have yielded high-resolution X-ray or electron diffraction data, opening the prospect of increased throughput for eukaryotic membrane protein structure determination. Here, we summarize the major expression systems available, and highlight technical advances that should facilitate more systematic screening of expression conditions for this physiologically important class of targets.

Domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation.

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

The quaternary structure of a calcium-permeable AMPA receptor: conservation of shape and symmetry across functionally distinct subunit assemblies.

AMPA receptors (AMPA-Rs) are formed as heterotetrameric combinations of subunits known as GluR1-GluR4. The calcium permeability of AMPA-Rs is controlled by the identity of the amino-acid side chain contributed by each subunit at a key position in the conductance pathway, which can be either a glutamine (Q) or an arginine (R). Tetramers assembled only from Q-containing subunits are calcium permeable. In contrast, tetramers that incorporate R-containing subunits are calcium impermeable. Both forms play key roles in physiological and pathophysiological processes in the central nervous system. Here, using electron microscopy, we present the first quaternary structure of a calcium-permeable Q-homomeric AMPA-R. The receptor is elongated, with overall 2-fold symmetry and a large central vestibule. It is thus similar to the structure previously reported for an AMPA-R assembled exclusively from R-subunits. Both structures differ from those reported for brain-derived but urea-washed "native" AMPA-Rs, which exhibited multiple asymmetrical conformations. However, even transient exposure of our Q-homomeric AMPA-Rs to urea significantly attenuates the binding of a conformationally specific antibody. As a result, we propose a model in which all AMPA-Rs share a 2-fold symmetrical structure and in which subunit-dependent differences in assembly, trafficking, and electrophysiology are mediated within the framework of fundamentally similar quaternary conformations.