Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively

The structural basis for Z α1-antitrypsin polymerization in the liver

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as “polymer” chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin

A translocation motif in relaxase TrwC specifically affects recruitment by its conjugative type IV secretion system

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation

The stressosome is the epicenter of the stress response in bacteria, and one of the largest bacterial nanomachines. How the stressosome integrates and transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT a kinase that is important for its activation. Here using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38Å resolution. The structure shows that RsbR and RsbS are organized in a 60 protomers truncated icosahedron. Two phosphorylation sites on RsbR (T175, T209) and one on RsbS (S56) are arranged on a horizontal row that is interrupted by a 13 amino acid flexible loop in RsbR. RsbR T175 and RsbS S56 are accessible on the surface and are phosphorylated under normal stress conditions. Access to T209 is partially hidden by the RsbR flexible loop, whose “open” or “closed” position, could modulate stressosome activation. Modification of the flexible loop or of residues involved in RsbR and RsbS interaction, results in a dominant negative phenotype. In addition, we showed that the interaction between three glutamic acids in the N terminal domain of RsbR and the membrane bound mini-protein Prli42 is essential for Listeria survival to stress. Taken together, our data provide the first atomic model of the stressosome core assembly, and highlight a loop that is important for stressosome activation, paving the way towards elucidating the structural basis of stressosome function in bacteria

Neutronenstreuexperimente zur mikroskopischen Dynamik in ungeordneten Polymersystemen

Adhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro

Structural characterisation of tissue-derived, disease-associated polymers of alpha-1-antitrypsin using conformation-selective antibodies and single-particle reconstructions from electron microscopy images

α1-Antitrypsin is an abundant plasma inhibitor of neutrophil elastase,expressed at high levels by hepatocytes, and one of the causative agents of a class of conformational diseases termed serpinopathies. In its active state, α1-antitrypsin is in a kinetically stable, but thermodynamically unstable, configuration, rendering it susceptible to inappropriate conformationalchange. In individuals homozygous for the Z (E342K) mutation, α1-antitrypsin accumulates in the liver as dense intracellular deposits, leading to a reduced level in circulation. These deposits are the consequence of an ‘ordered aggregation’ that yields linear, unbranched protein chains, termed polymers, that are both extremely stable and functionally inactive. The circulating deficiency results in a protease-antiprotease imbalance in the lung, predisposing affected individuals to emphysema and COPD, whilst the hepatic accumulation can lead to liver disease, including cirrhosis and hepatocellular carcinoma. Our aim is to define the molecular details of the polymerisation pathway, in which α1-antitrypsin passes through different conformational states as it transitions from the active monomer via one or more structural intermediates to a hyperstable polymeric form. Different models have been proposed for the terminal structure adopted by the pathological polymer; these are are largely based on characterisation of polymers produced under conditions mechanistically or biologically distinct from those existing in vivo, and as such their relevance to the pathological context has not been established. To probe the structural and energetic aspects of the polymerisation pathway, we have generated a molecular toolkit of conformation-specific monoclonal antibodies (mAbs), and mapped their epitopes. We have utilised these mAbs and applied single-particle reconstruction techniques to negative stain and cryo-EM images of polymers extracted from patient explant liver tissue. The resulting maps, in conjunction with molecular modelling, have allowed us to critically evaluate the proposed mechanisms of polymer formation

Structure of a type IV secretion system

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells1, 2, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells3. Given the complex choreography of the substrate through the secretion apparatus4, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems

Cryo-EM structure of a type IV secretion system

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili

The structural basis for Z α₁-antitrypsin polymerization in the liver

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin

The structural basis for Z α₁-antitrypsin polymerization in the liver

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α$_1$-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α$_1$-antitrypsin as “polymer” chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α$_1$-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α$_1$-antitrypsin