Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.

Penicillin-Binding Proteins (PBPs) and Bacterial Cell Wall Elongation Complexes.

The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other ß-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.

Structural characterization of the sporulation protein GerM from Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis responds to starvation by entering a morphological differentiation process leading to the formation of a highly resistant spore. Early in the sporulation process, the cell asymmetrically divides into a large compartment (the mother cell) and a smaller one (the forespore), which will maturate into a resistant spore. Proper development of the forespore requires the assembly of a multiprotein complex called the SpoIIIA-SpoIIQ complex or "A-Q complex". This complex involves the forespore protein SpoIIQ and eight mother cell proteins (SpoIIIAA to SpoIIIAH), many of which share structural similarities with components of specialized secretion systems and flagella found in Gram-negative bacteria. The assembly of the A-Q complex across the two membranes that separate the mother cell and forespore was recently shown to require GerM. GerM is a lipoprotein composed of two GerMN domains, a family of domains with unknown function. Here, we report X-ray crystallographic structures of the first GerMN domain of GerM at 1.0 Šresolution, and of the soluble domain of GerM (the tandem of GerMN domains) at 2.1 Šresolution. These structures reveal that GerMN domains can adopt distinct conformations and that the core of these domains display structural similarities with ring-building motifs found in components of specialized secretion system and in SpoIIIA proteins. This work provides an additional piece towards the structural characterization of the A-Q complex.

Substitutions in PBP2b from ß-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity.

Pneumococcus resists ß-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because ß-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different ß-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism.

Crystal structure of pb9, the distal tail protein of bacteriophage T5: a conserved structural motif among all siphophages.

The tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.

Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex.

The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization.

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.

Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with ß-Lactones.

ß-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to ß-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of ß-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four ß-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the ß-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

MagC is a NplC/P60-like member of the α-2-macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan.

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Self-association of MreC as a regulatory signal in bacterial cell wall elongation.

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.

Sortase-mediated pilus fiber biogenesis in Streptococcus pneumoniae.

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.

Penicillin-binding proteins and beta-lactam resistance.

A number of ways and means have evolved to provide resistance to eubacteria challenged by beta-lactams. This review is focused on pathogens that resist by expressing low-affinity targets for these antibiotics, the penicillin-binding proteins (PBPs). Even within this narrow focus, a great variety of strategies have been uncovered such as the acquisition of an additional low-affinity PBP, the overexpression of an endogenous low-affinity PBP, the alteration of endogenous PBPs by point mutations or homologous recombination or a combination of the above.

Bacterial secretins: Mechanisms of assembly and membrane targeting.

Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.

Tandem use of X-ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38-kDa apolipoprotein.

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal residues, are systematically absent from eukaryotic genome databases. As a consequence, HPBP amino acids sequence had to be first assigned from the electronic density map. Then, an original approach combining X-ray crystallography and mass spectrometry provides the complete and a priori exact sequence of the 38-kDa HPBP. This first complete sequence of a eukaryotic DING protein will be helpful to study HPBP and the entire DING protein family.

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase.

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.

Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate.

Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP.

Serendipitous discovery and X-ray structure of a human phosphate binding apolipoprotein.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The systematic absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation between genes belonging to evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the only known transporter capable of binding phosphate ions in human plasma and may become a new predictor of or a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.