An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

SAS-Based Structural Modelling and Model Validation.

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.

Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.

Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease.

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.

Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.

Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly.

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.

Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a ß-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure-function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

Protein interactions in 3D: from interface evolution to drug discovery.

Over the past 10years, much research has been dedicated to the understanding of protein interactions. Large-scale experiments to elucidate the global structure of protein interaction networks have been complemented by detailed studies of protein interaction interfaces. Understanding the evolution of interfaces allows one to identify convergently evolved interfaces which are evolutionary unrelated but share a few key residues and hence have common binding partners. Understanding interaction interfaces and their evolution is an important basis for pharmaceutical applications in drug discovery. Here, we review the algorithms and databases on 3D protein interactions and discuss in detail applications in interface evolution, drug discovery, and interface prediction.

MeMotif: a database of linear motifs in alpha-helical transmembrane proteins.

Membrane proteins are important for many processes in the cell and used as main drug targets. The increasing number of high-resolution structures available makes for the first time a characterization of local structural and functional motifs in alpha-helical transmembrane proteins possible. MeMotif (http://projects.biotec.tu-dresden.de/memotif) is a database and wiki which collects more than 2000 known and novel computationally predicted linear motifs in alpha-helical transmembrane proteins. Motifs are fully described in terms of several structural and functional features and editable. Motifs contained in MeMotif can be used in different biological applications, from the identification of biochemically important functional residues which are candidates for mutagenesis experiments to the improvement of tools for transmembrane protein modeling.

Structural modeling of histone methyltransferase complex Set1C from Saccharomyces cerevisiae using constraint-based docking.

Set1C is a histone methyltransferase playing an important role in yeast gene regulation. Modeling the structure of this eight-subunit protein complex is an important open problem to further elucidate its functional mechanism. Recently, there has been progress in modeling of larger complexes using constraints to restrict the combinatorial explosion in binary docking of subunits. Here, we model the subunits of Set1C and develop a constraint-based docking approach, which uses high-quality protein interaction as well as functional data to guide and constrain the combinatorial assembly procedure. We obtained 22 final models. The core complex consisting of the subunits Set1, Bre2, Sdc1 and Swd2 is conformationally conserved in over half of the models, thus, giving high confidence. We characterize these high-confidence and the lower confidence interfaces and discuss implications for the function of Set1C.

Structural fragment clustering reveals novel structural and functional motifs in alpha-helical transmembrane proteins.

BACKGROUND: A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. RESULTS: We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to alpha-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94%) appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1) a dimer interface motif found in voltage-gated chloride channels, (2) a proton transfer motif found in heme-copper oxidases, and (3) a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. CONCLUSIONS: Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.

Molecular Organization of Soluble Type III Secretion System Sorting Platform Complexes.

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components are unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology.

The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase.

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a(3)/Cu(B) site, and for proton pumping. When present, the side chain of Glu-242 is orientated "down" towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity "above" Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an "up" position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the "up" position is preferred energetically when the cavity contains four water molecules, but the "down" position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the "resting" state of the enzyme, and that the "up/down" equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O(2) reduction.

The K-pathway revisited: a computational study on cytochrome c oxidase.

Cytochrome c oxidase contains two established proton-conducting structures, the D- and K-pathways. The role of the K-pathway appears to be to conduct the first two protons to be used in water formation, which are taken up on reduction of the oxidized enzyme. Previous computational work has suggested that Lys(I)-319 is neutral over a large pH range and in various redox states. We have constructed oxidase models in different redox states using quantum-chemically derived charge parameters for the redox metal centers. The protonation behaviour of titratable sites in the two-subunit enzyme was defined by continuum electrostatics. The calculations reported here show substantial protonation of Lys(I)-319 at neutral pH once the stable X-ray crystallographic water molecule found immediately next to it is treated explicitly. The immediate structure of the Lys(I)-319 environment is independent of redox state, but the pK(a) value of this residue changes with the redox state of the binuclear heme a3/Cu(B) site whenever that change is electrically uncompensated. Lys(I)-319 is also found to interact electrostatically with the conserved residue Glu(II)-62 in subunit II. These results are discussed in relation to the role of the K-pathway in oxidase function.

Progress in small-angle scattering from biological solutions at high-brilliance synchrotrons.

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.

Resolution of ab initio shapes determined from small-angle scattering.

Spatial resolution is an important characteristic of structural models, and the authors of structures determined by X-ray crystallography or electron cryo-microscopy always provide the resolution upon publication and deposition. Small-angle scattering of X-rays or neutrons (SAS) has recently become a mainstream structural method providing the overall three-dimensional structures of proteins, nucleic acids and complexes in solution. However, no quantitative resolution measure is available for SAS-derived models, which significantly hampers their validation and further use. Here, a method is derived for resolution assessment for ab initio shape reconstruction from scattering data. The inherent variability of the ab initio shapes is utilized and it is demonstrated how their average Fourier shell correlation function is related to the model resolution. The method is validated against simulated data for proteins with known high-resolution structures and its efficiency is demonstrated in applications to experimental data. It is proposed that henceforth the resolution be reported in publications and depositions of ab initio SAS models.