Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae

Abstract RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist Sp RecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism

Using Cryo-EM to Investigate Bacterial Secretion Systems

International audienceBacterial secretion systems are responsible for releasing macromolecules to the extracellular milieu or directly into other cells. These membrane complexes are associated with pathogenicity and bacterial fitness. Understanding of these large assemblies has exponentially increased in the last few years thanks to electron microscopy. In fact, a revolution in this field has led to breakthroughs in characterizing the structures of secretion systems and other macromolecular machineries so as to obtain high-resolution images of complexes that could not be crystallized. In this review, we give a brief overview of structural advancements in the understanding of secretion systems, fo-cusing in particular on cryo-electron microscopy, whether tomography or single-particle analysis. We describe how such techniques have contributed to knowledge of the mechanism of macromolecule secretion in bacteria and the impact they will have in the future

Binding mechanisms of therapeutic antibodies to human CD20

International audienceMonoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo–electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20

Filamentation of the bacterial bi-functional alcohol/aldehyde dehydrogenase AdhE is essential for substrate channeling and enzymatic regulation

International audienceAcetaldehyde-alcohol dehydrogenase (AdhE) enzymes are a key metabolic enzyme in bacterial physiology and pathogenicity. They convert acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in an anaerobic environment. This two-step reaction is associated to NAD + regeneration, essential for glycolysis. The bifunctional AdhE enzyme is conserved in all bacterial kingdoms but also in more phylogenetically distant microorganisms such as green microalgae. It is found as an oligomeric form called spiro-somes, for which the function remains elusive. Here, we use cryo-electron microscopy to obtain structures of Escherichia coli spirosomes in different conformational states. We show that spirosomes contain active AdhE monomers, and that AdhE filamentation is essential for its activity in vitro and function in vivo. The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling within the filament and for the regulation of enzyme activity

Structural insights into the mechanism of DNA branch migration during homologous recombination in bacteria

International audienceSome DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

International audienceBacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii , Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell