Structure of a translocation signal domain mediating conjugative transfer by Type IV secretion systems

Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three sub-domains with striking structural homology with helicase sub-domains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities

An activation domain of plasmid R1 TraI protein delineates stages of gene transfer initiation

Bacterial conjugation is a form of type IV secretion that transports protein and DNA to recipient cells. Specific bacteriophage exploit the conjugative pili and cell envelope spanning protein machinery of these systems to invade bacterial cells. Infection by phage R17 requires F-like pili and coupling protein TraD, which gates the cytoplasmic entrance of the secretion channel. Here we investigate the role of TraD in R17 nucleoprotein uptake and find parallels to secretion mechanisms. The relaxosome of IncFII plasmid R1 is required. A ternary complex of plasmid oriT, TraD and a novel activation domain within the N-terminal 992 residues of TraI contributes a key mechanism involving relaxase-associated properties of TraI, protein interaction and the TraD ATPase. Helicase-associated activities of TraI are dispensable. These findings distinguish for the first time specific protein domains and complexes that process extracellular signals into distinct activation stages in the type IV initiation pathway. The study also provided insights into the evolutionary interplay of phage and the plasmids they exploit. Related plasmid F adapted to R17 independently of TraI. It follows that selection for phage resistance drives not only variation in TraA pilins but diversifies TraD and its binding partners in a plasmid-specific manner

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations

Structure of the Bacterial Sex F Pilus reveals an assembly of a Stoichiometric Protein-Phospholipid Complex

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection

Structure of a Chaperone-Usher Pilus reveals the molecular basis of rod uncoiling

Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion

NMR studies on protein - protein interactions participating in regulatory mechanisms in conjugative DNA transfer

Protein-Protein Interaktionen (PPIs) spielen eine entscheidende Rolle in biologischen Prozessen. Diese spezifischen Wechselwirkungen bilden auch die Grundlage für die Entwicklung von molekularen Inhibitoren, etwa gegen die Ausbreitung von Antibiotika-Resistenzen durch bakterielle Konjugation. Ziel dieser Studie war die Beschreibung von Protein-Protein Interaktionen der bakteriellen DNA-Übertragung.Dabei wurden eventuelle Wechselwirkungen zwischen TraM, einem Hilfsprotein, und der Relaxase/Helikase TraI studiert. Weiters wurden die Interaktionen zwischen TraD und TraI betrachtet. Das Protein ParM wurde hinsichtlich seines DNA-Bindungsverhalten und seiner Wechselwirkungen mit anderen Relaxosom-Proteinen charakterisiert. Die durchgeführten Experimente zeigen beispielsweise, dass TraI und TraM nicht über deren N-Terminus wechselwirken, obwohl eine Interaktion mittels des C-Terminus nicht ausgeschlossen werden kann. Weiters wurde die erste Struktur eines Translokations-Proteins (TraI 570-795) anhand der Röntgen-Kristallographie gelöst. Dabei zeigen sich strukturelle Ähnlichkeiten zum Protein RecD2, das zur SF1 Familie gehört. Zwei SH3-ähnliche Domänen weisen dabei auf eine Signaltransduktionsfunktion hin. Letztendlich konnte gezeigt werden, dass TraI direkt mit TraD wechselwirkt und dies den zugrundeliegenden Mechanismus für T4S Kanalaktivierung darstellt. Außerdem gibt die Struktur Hinweise darauf, dass ParM mit TraI wechselwirken könnte.Zusammengefasst gewähren die erzielten Ergebnisse Einblicke in den Prozess der bakteriellen Konjugation.In this study the main goal was to define the protein ? protein interactions (PPI?s) which are necessary for regulation and signaling mechanisms in conjugal DNA transfer in bacteria. The role of the possible interaction between the auxiliary protein TraM and the relaxase ? helicase protein TraI was investigated. Next, the mechanism of T4S channel activation and substrate selection was considered to be driven by simple interactions between coupling protein TraD and TraI, via its translocation domain (TS). Eventually the role of partitioning actin ? like protein ParM in DNA processing was tested if it might stimulate the generation of nicked DNA because it is part of the relaxosome by interacting with TraI or other relaxosome component. The second hypothesis tested was if ParM is responsible for positioning the relaxosome to the T4S system and pass it by to the TraD via interaction mechanism.The experiments performed during this study have shown that the possible interactions between TraI and TraM are not involving the N-terminally encoded relaxase domain. However interactions including the C ?terminally encoded helicase domain cannot be excluded. The structure of the first translocation signal (TraI 570-795) has been solved using X-ray crystallography. It shows a partial homology to the structure of the member of SF1 family of helicases ? RecD2. Two duplications of 2B domains can be found. These SH3 like domain folds, are likely to be essential for signaling function in eukaryotic systems. Our study shows that TraD is interacting with TraI by this specific domain, indicating that this is the mechanism for T4S channel activation.Eventually, the collected dataset indicates that the protein which might interact with ParM is TraI and if that is the case, then the interaction happens also via the TSA domainAll together, the data gave more valuable information to understand the mechanism of substrates secretion during bacterial conjugation.vorgelegt von Adam RedzejZsfassung in dt. und engl. SpracheGraz, Univ., Diss., 2012OeBB(VLID)22240

Studies towards enzymatic kinetic resolutions of 1,3-diol peptidomimetics obtained via the Ugi reaction

This paper is dedicated to Prof. Janusz Lipkowski on the occasion of his 70th birthday Enzymatic methods in combination with the multicomponent Ugi condensation make a very efficient method for simple synthesis of non-racemic peptidomimetics. The aim of the studies was to develop an enzymatic kinetic resolution of 1,3-diol peptidomimetics providing nonracemic compounds. Among many applications, 1,3-diols can serve as intermediates in the synthesis of anticancer agents- β-acyloxymethacrylic amides. Stereoselective enzymatic acylation and hydrololysis of Ugi products were investigated. The enantiomeric or diastereomeric excesses were determined in both cases. As a result, an efficient enzymatic method for the synthesis chiral, non-racemic 1,3-diol peptidomimetics was developed

Structural Analysis of Protein Complexes by Cryo-Electron Microscopy

Structural studies of bio-complexes using single particle cryo-Electron Microscopy (cryo-EM) is nowadays a well-established technique in structural biology and has become competitive with X-ray crystallography. Development of digital registration systems for electron microscopy images and algorithms for the fast and efficient processing of the recorded images and their following analysis has facilitated the determination of structures at near-atomic resolution. The latest advances in EM have enabled the determination of protein complex structures at 1.4-3 Å resolution for an extremely broad range of sizes (from ~100 kDa up to hundreds of MDa (Bartesaghi et al., Science 348(6239):1147-1151, 2015; Herzik et al., Nat Commun 10:1032, 2019; Wu et al., J Struct Biol X 4:100020, 2020; Zhang et al., Nat Commun 10:5511, 2019; Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020; https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year )). In 2022, nearly 1200 structures deposited to the EMDB database were at a resolution of better than 3 Å ( https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year ).To date, the highest resolutions have been achieved for apoferritin, which comprises a homo-oligomer of high point group symmetry (O432) and has rigid organization together with high stability (Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020). It has been used as a test object for the assessments of modern cryo-microscopes and processing methods during the last 5 years. In contrast to apoferritin bacterial secretion systems are typical examples of multi protein complexes exhibiting high flexibility owing to their functions relating to the transportation of small molecules, proteins, and DNA into the extracellular space or target cells. This makes their structural characterization extremely challenging (Barlow, Methods Mol Biol 532:397-411, 2009; Costa et al., Nat Rev Microbiol 13:343-359, 2015). The most feasible approach to reveal their spatial organization and functional modification is cryo-electron microscopy (EM). During the last decade, structural cryo-EM has become broadly used for the analysis of the bio-complexes that comprise multiple components and are not amenable to crystallization (Lyumkis, J Biol Chem 294:5181-5197, 2019; Orlova and Saibil, Methods Enzymol 482:321-341, 2010; Orlova and Saibil, Chem Rev 111(12):7710-7748, 2011).In this review, we will describe the basics of sample preparation for cryo-EM, the principles of digital data collection, and the logistics of image analysis focusing on the common steps required for reconstructions of both small and large biological complexes together with refinement of their structures to nearly atomic resolution. The workflow of processing will be illustrated by examples of EM analysis of Type IV Secretion System

Studies toward Novel Peptidomimetic Inhibitors of Thioredoxin–Thioredoxin Reductase System

Thioredoxins (Trx) are ubiquitous multifunctional low-molecular weight proteins that together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis in NADPH-dependent reactions. An increasing number of data reveal that the Trx–TrxR system is an attractive target for anticancer therapies. In this work, we have elaborated a new and simple synthetic approach employing Ugi reaction to synthesize several new inhibitors of this system. The influence of various electrophilic fragments of this new class of compounds on the inhibition of the Trx–TrxR system was evaluated. As a result, a new compound <b>19a</b> (SK053), which inhibits the activity of the Trx–TrxR system and exhibits antitumor activity, was obtained. Biologic analyses revealed that <b>19a</b> inhibits induction of NF-κB and AP-1 and decreases H₂O₂ scavenging capacity in tumor cells. Altogether, we show that <b>19a</b> is a novel potential antitumor peptidomimetic inhibitor that can be used as a starting compound for further optimization

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