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Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains., Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes assembling complex natural products. Here, the structures of a Thermobifida fusca NRPS condensation domain bound to the substrate-bearing peptidyl carrier protein (PCP) domain provide insight into the mechanisms of substrate selectivity and engagement within the catalytic pocket.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420., F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine- N -acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.

Covering: up to July 2018 Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. By decoupling peptide synthesis from the ribosome, NRPS assembly lines are able to access a significant pool of amino acid monomers for peptide synthesis. This is combined with a modular protein architecture that allows for great variation in stereochemistry, peptide length, cyclisation state and further modifications. The architecture of NRPS assembly lines relies upon a repetitive set of catalytic domains, which are organised into modules responsible for amino acid incorporation. Central to NRPS-mediated biosynthesis is the carrier protein (CP) domain, to which all intermediates following initial monomer activation are bound during peptide synthesis up until the final handover to the thioesterase domain that cleaves the mature peptide from the NRPS. This mechanism makes understanding the protein-protein interactions that occur between different NRPS domains during peptide biosynthesis of crucial importance to understanding overall NRPS function. This endeavour is also highly challenging due to the inherent flexibility and dynamics of NRPS systems. In this review, we present the current state of understanding of the protein-protein interactions that govern NRPS-mediated biosynthesis, with a focus on insights gained from structural studies relating to CP domain interactions within these impressive peptide assembly lines.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape., eLife digest Many bacteria are surrounded by both a cell membrane and a cell wall – a rigid outer covering made of sugars and short protein chains. The cell wall often determines which of a variety of shapes – such as rods or spheres – the bacteria grow into. One protein required to form the rod shape is called MreB. This protein forms filaments that bind to the bacteria’s cell membrane and associate with the enzymes that build the cell wall. Together, these filament-enzyme complexes rotate around the cell to build and reinforce the cell wall in a hoop-like manner. But how do the MreB filaments know how to move around the circumference of the rod, instead of moving in any other direction? Using a technique called total internal reflection microscopy to study how MreB filaments move across bacteria cells, Hussain, Wivagg et al. show that the filaments sense the shape of a bacterium by orienting along the direction of greatest curvature. As a result, the filaments in rod-shaped cells orient and move around the rod, while in spherical bacteria they move in all directions. However, spherical bacteria can regenerate into rods from small surface ‘bulges’. The MreB filaments in the bulges move in an oriented way, helping them to generate the rod shape. Hussain, Wivagg et al. also found that forcing cells that lack a cell wall into a rod shape caused the MreB filaments bound to the cell membrane to orient and circle around the rod. This shows that the organization of the filaments is sufficient to shape the cell wall. In the future, determining what factors control the activity of the MreB filaments and the enzymes they associate with might reveal new targets for antibiotics that disrupt the cell wall and so kill the bacteria. This will require higher resolution microscopes to be used to examine the cell wall in more detail. The activity of all the proteins involved in building cell walls will also need to be extensively characterized.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape ofBacillus subtilis.MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomesin vitro,orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

Highlights • Crystal structure of crenactin solved in polymeric form at 3.2 Å resolution. • Crenactin is closely related to eukaryotic actin, RMSD 1.6 Å. • Crenactin forms right-handed filaments with 8 subunits per complete turn. • Rare crystal structure of helical filament of actin-like protein., Polymerising proteins of the actin family are nearly ubiquitous. Crenactins, restricted to Crenarchaea, are more closely related to actin than bacterial MreB. Crenactins occur in gene clusters hinting at an unknown, but conserved function. We solved the crystal structure of crenactin at 3.2 Å resolution. The protein crystallises as a continuous right-handed helix with 8 subunits per complete turn, spanning 419 Å. The structure of crenactin shows several loops that are longer than in actin, but overall, crenactin is closely related to eukaryotic actin, with an RMSD of 1.6 Å. Crenactin filaments imaged by electron microscopy showed polymers with very similar helical parameters.

In Gram-negative bacteria, the bacterial cell wall biosynthetic mechanism requires the coordinated action of enzymes and structural proteins located in the cytoplasm, within the membrane, and in the periplasm of the cell. Its main component, peptidoglycan (PG), is essential for cell division and wall elongation. Penicillin-binding proteins (PBPs) catalyze the last steps of PG biosynthesis, namely the polymerization of glycan chains and the cross-linking of stem peptides, and can be either monofunctional or bifunctional. Their action is coordinated with that of other enzymes essential for cell-wall biosynthesis, such as lytic transglycosylases (LT). Here, we have studied SltB1, an LT from Pseudomonas aeruginosa, and identified that it forms a complex with PBP2, a monofunctional enzyme, which requires the presence of Ca(2+). In addition, we have solved the structure of SltB1 to a high resolution, and identified that it harbors an EF-hand like motif containing a Ca(2+) ion displaying bipyramidal coordination. These studies provide initial structural details that shed light on the interactions between the PG biosynthesis enzymes in P. aeruginosa.

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

SummaryPili are fibrous virulence factors associated directly to the bacterial surface that play critical roles in adhesion and recognition of host cell receptors. The human pathogen Streptococcus pneumoniae carries a single pilus-related adhesin (RrgA) that is key for infection establishment and provides protection from bacterial challenge in animal infection models, but details of these roles remain unclear. Here we report the high-resolution crystal structure of RrgA, a 893-residue elongated macromolecule whose fold contains four domains presenting both eukaryotic and prokaryotic origins. RrgA harbors an integrin I collagen-recognition domain decorated with two inserted “arms” that fold into a positively charged cradle, as well as three “stalk-forming” domains. We show by site-specific mutagenesis, mass spectrometry, and thermal shift assays that intradomain isopeptide bonds play key roles in stabilizing RrgA's stalk. The high sequence similarity between RrgA and its homologs in other Gram-positive microorganisms suggests common strategies for ECM recognition and immune evasion.

Pili are surface-linked virulence factors that play key roles in infection establishment in a variety of pathogenic species. In Gram-positive pathogens, pilus formation requires the action of sortases, dedicated transpeptidases that covalently associate pilus building blocks. In Streptococcus pneumoniae, a major human pathogen, all genes required for pilus formation are harbored in a single pathogenicity islet which encodes three structural proteins (RrgA, RrgB, RrgC) and three sortases (SrtC-1, SrtC-2, SrtC-3). RrgB forms the backbone of the streptococcal pilus, to which minor pilins RrgA and RrgC are covalently associated. SrtC-1 is the main sortase involved in polymerization of the RrgB fiber and displays a lid which encapsulates the active site, a feature present in all pilus-related sortases. In this work, we show that catalysis by SrtC-1 proceeds through a catalytic triad constituted of His, Arg, and Cys and that lid instability affects protein fold and catalysis. In addition, we show by thermal shift analysis that lid flexibility can be stabilized by the addition of substrate-like peptides, a feature shared by other periplasmic transpeptidases. We also report the characterization of a trapped acyl-enzyme intermediate formed between SrtC-1 and RrgB. The presence of lid-encapsulated sortases in the pilus biogenesis systems in many Gram-positive pathogens points to a common mechanism of substrate recognition and catalysis that should be taken into consideration in the development of sortase inhibitors.

Summary Streptococcus pneumoniae is a piliated pathogen whose ability to circumvent vaccination and antibiotic treatment strategies is a cause of mortality worldwide. Pili play important roles in pneumococcal infection, but little is known about their biogenesis mechanism or the relationship between components of the pilus-forming machinery, which includes the fiber pilin (RrgB), two minor pilins (RrgA, RrgC), and three sortases (SrtC-1, SrtC-2, SrtC-3). Here we show that SrtC-1 is the main pilus-polymerizing transpeptidase, and electron microscopy analyses of RrgB fibers reconstituted in vitro reveal that they structurally mimic the pneumococcal pilus backbone. Crystal structures of both SrtC-1 and SrtC-3 reveal active sites whose access is controlled by flexible lids, unlike in non-pilus sortases, and suggest that substrate specificity is dictated by surface recognition coupled to lid opening. The distinct structural features of pilus-forming sortases suggest a common pilus biogenesis mechanism that could be exploited for the development of broad-spectrum antibacterials.

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: http://dx.doi.org/10.7554/eLife.02634.001, eLife digest Bacterial cells come in a variety of different shapes, including spheres, rods, spirals, and crescents. Shape is important for bacterial cells because it plays a role in cell division, helps to maximize the uptake of nutrients, and aids cell movement. The shape of a cell is determined mainly by its cytoskeleton, a form of ‘scaffolding’ within the cell that is made of various protein filaments. The bacterial cytoskeleton was discovered over 20 years ago, but it has not been studied as much as the cytoskeletons of yeast, plant, animals, and other eukaryotes. Many of the bacterial proteins and filaments that make up the cytoskeleton are similar to those found in eukaryotes. A protein called MreB, for example, is the bacterial equivalent of actin, which performs a wide range of roles in eukaryotes. However, van den Ent, Izoré et al. have now shown that the detailed structure of MreB filaments is different to that of actin filaments. It has been known for some time that actin filaments are composed of two strands of actin proteins that are twisted and parallel with each other. MreB filaments are also made of two strands of MreB proteins, but van den Ent, Izoré et al. found that these strands are straight, not twisted, and that they are antiparallel rather than parallel. Thus, unlike other filaments of actin-like proteins, where the two ends of the filament are clearly different from each other, the antiparallel strands of MreB form a double filament without a clear direction. van den Ent, Izoré et al. also showed that MreB double filaments can bind to surfaces that mimic a bacterial cell membrane, and that mutant bacterial cells without these double filaments adopt the wrong cell shape. Further experiments exposed potential targets on the MreB filaments for antibiotics that could treat bacterial infections. DOI: http://dx.doi.org/10.7554/eLife.02634.002

International audience; Pseudomonas aeruginosa is an opportunistic human pathogen that employs a finely tuned type III secretion system (T3SS) to inject toxins directly into the cytoplasm of target cells. ExsB is a 15.6-kDa protein encoded in a T3SS transcription regulation operon that displays high sequence similarity to YscW, a lipoprotein from Yersinia spp. whose genetic neighborhood also involves a transcriptional regulator, and has been shown to play a role in the stabilization of the outer membrane ring of the T3SS. Here, we show that ExsB is expressed in P. aeruginosa upon induction of the T3SS, and subcellular fractionation studies reveal that it is associated with the outer membrane. The high-resolution crystal structure of ExsB shows that it displays a compact β-sandwich fold with interdependent β-sheets. ExsB possesses a large patch of basic residues that could play a role in membrane recognition, and its structure is distinct from that of MxiM, a lipoprotein involved in secretin stabilization in Shigella, as well as from those of Pil lipoproteins involved in pilus biogenesis. These results reveal that small lipoproteins involved in formation of the outer membrane secretin ring display clear structural differences that may be related to the different functions they play in these systems.

International audience; The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

Pseudomonas ΦKZ-like bacteriophages encode a group of related tubulin/FtsZ-like proteins believed to be essential for the correct centring of replicated bacteriophage virions within the bacterial host. In this study, we present crystal structures of the tubulin/FtsZ-like protein TubZ from Pseudomonas bacteriophage ΦKZ in both the monomeric and protofilament states, revealing that ΦKZ TubZ undergoes structural changes required to polymerise, forming a canonical tubulin/FtsZ-like protofilament. Combining our structures with previous work, we propose a polymerisation–depolymerisation cycle for the Pseudomonas bacteriophage subgroup of tubulin/FtsZ-like proteins. Electron cryo-microscopy of ΦKZ TubZ filaments polymerised in vitro implies a long-pitch helical arrangement for the constituent protofilaments. Intriguingly, this feature is shared by the other known subgroup of bacteriophage tubulin/FtsZ-like proteins from Clostridium species, which are thought to be involved in partitioning the genomes of bacteriophages adopting a pseudo-lysogenic life cycle., Graphical abstract Highlights ► Bacteriophage ΦKZ encodes a tubulin/FtsZ homologue, TubZ, similar to 201Φ2-1 PhuZ. ► We have resolved crystal structures of ΦKZ TubZ as both a monomer and a protofilament. ► ΦKZ TubZ undergoes structural changes only at its surface during polymerisation. ► Electron microscopy shows that ΦKZ TubZ forms intertwined helical filaments in vitro. ► This feature is common to other TubZs in Bacillus plasmids and Clostridium prophage.