Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.

Exploring Host-Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Modeling Flexible Protein Structure With AlphaFold2 and Crosslinking Mass Spectrometry.

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2 and conformer selection using XL-MS data. For conformer selection, we developed two scores-the monolink probability score (MP) and the crosslink probability score (XLP)-both of which are based on residue depth from the protein surface. We benchmarked MP and XLP on a large dataset of decoy protein structures and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein, first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles-or a conformation within 1 Å of this model-was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of glutamine-binding periplasmic protein, and these results were further improved by including the "occupancy" of the monolinks. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models. The MP and XLP scoring functions mentioned above are available at https://gitlab.com/topf-lab/xlms-tools.

Residues within the LptC transmembrane helix are critical for Escherichia coli LptB2 FG ATPase regulation.

Lipopolysaccharide (LPS) synthesis in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB2 FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand-off of LPS from LptB2 FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2 FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2 FGC complex and regulate the ATPase activity of LptB.

The structure of MucD from Pseudomonas syringae revealed N-terminal loop-mediated trimerization of HtrA-like serine protease.

Protein quality control mechanisms are essential for maintaining cellular integrity, and the HtrA family of serine proteases plays a crucial role in handling folding stress in prokaryotic periplasm. Escherichia coli harbors three HtrA members, namely, DegS, DegP, and DegQ, which share a common domain structure. MucD, a putative HtrA family member that resembles DegP, is involved in alginate biosynthesis regulation and the stress response. Pseudomonas syringae causes plant diseases and opportunistic infections in humans. This study presents the high-resolution structure of MucD from Pseudomonas syringae (psMucD), revealing its composition as a typical HtrA family serine protease with protease and PDZ domains. Its findings suggest that psMucD containing one PDZ domain is a trimer in solution, and psMucD trimerization is mediated by its N-terminal loop. Sequence and structural analyses revealed similarities and differences with other HtrA family members. Additionally, this study provides a model of psMucD's catalytic process, comparing it with other members of the HtrA family of serine proteases.

Approaches for high-throughput quantification of periplasmic recombinant proteins.

The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development.

Identification of enzymatic functions of osmo-regulated periplasmic glucan biosynthesis proteins from Escherichia coli reveals a novel glycoside hydrolase family.

Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Pathogenicity of many pathogens is lost by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this study, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are ß-1,2-glucanases with remarkably different activity from each other, establishing a new glycoside hydrolase family, GH186. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway among glycoside hydrolase families is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family.

Expression and purification of Drosophila OBP44a with the aids of LC-MS and NMR.

The production of highly purified native soluble proteins in large quantities is crucial for studying protein structure and function. Odorant binding proteins (OBPs) are small, soluble, extracellular proteins with multiple disulfide bonds, whose functions include, but are not limited to, binding hydrophobic molecules and delivering them to their corresponding receptors expressed on insect olfactory receptor neurons. Expression of proteins with multiple disulfide bonds like OBPs usually results in insolubility and low yield, which has been a significant barrier to understanding their biological roles and physiological functions. In the E. coli system, expression of OBPs often results in insoluble inclusion bodies or a limited amount of periplasmic soluble proteins. Although expression of OBPs in eukaryotic systems such as Sf9 insect cells or yeast Pichia pastoris can increase the solubility of the protein, the process remains insufficient. Additionally, monitoring the purity and native apo state of the protein is critical for establishing the correct conformation of the protein. In this study, we employed an E. coli host with an altered intracellular environment to produce cytosolic soluble OBP44a protein, which yielded over 100 mg/L. We monitored the integrity of disulfide bonds throughout the purification process using LC-MS and used NMR to ensure the final product adopted a single conformation. Our study presents an efficient method for obtaining large quantities of soluble proteins in a single conformation, which enables extensive in vitro studies of secreted proteins like OBPs.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

Flexible Client-Dependent Cages in the Assembly Landscape of the Periplasmic Protease-Chaperone DegP.

The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of density maps and structural models that include those for novel particles of approximately 30 and 60 monomers. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.

Spy&IAC enables specific capture of SpyTagged proteins for rapid assembly of plug-and-display nanoparticle vaccines.

From modular vaccine production to protein assembly on nanoparticles, the SpyCatcher/SpyTag system provides a convenient plug-and-display procedure. Here, we established a general-purpose immunoaffinity chromatography (IAC) method for SpyTagged proteins (Spy&IAC). SpyTags are displayed on the surface of nanoparticles to induce high-affinity monoclonal antibodies, allowing the specific capture of the target protein. Taking the key core antigenic regions of two coronaviruses that are currently more threatened in the field of human and animal diseases, the nucleocapsid (N) protein of SARS-CoV-2 and the COE protein of porcine epidemic diarrhea virus (PEDV) as model proteins, a purification model with SpyTag at the N-terminal or C-terminal expressed in E. coli or mammalian cells was constructed. After the efficient elution of Spy&IAC, the final yield of several proteins is about 3.5-15 mg/L culture, and the protein purity is above 90 %. Purification also preserves the assembly function and immunogenicity of the protein to support subsequent modular assembly and immunization programs. This strategy provides a general tool for the efficient purification of SpyTagged proteins from different expression sources and different tag positions, enabling the production of modular vaccines at lower cost and in a shorter time, which will prepare the public health field for potential pandemic threats.

An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene - lamA - leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell - the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA. Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry.

Methionine oxidation under anaerobic conditions in Escherichia coli.

Repairing oxidative-targeted macromolecules is a central mechanism necessary for living organisms to adapt to oxidative stress. Reactive oxygen and chlorine species preferentially oxidize sulfur-containing amino acids in proteins. Among these amino acids, methionine can be converted into methionine sulfoxide. This post-translational oxidation can be reversed by methionine sulfoxide reductases, Msr enzymes. In Gram-negative bacteria, the antioxidant MsrPQ system is involved in the repair of periplasmic oxidized proteins. Surprisingly, in this study, we observed in Escherichia coli that msrPQ was highly expressed in the absence of oxygen. We have demonstrated that the anaerobic induction of msrPQ was due to chlorate (ClO3 - ) contamination of the Casamino Acids. Molecular investigation led us to determine that the reduction of chlorate to the toxic oxidizing agent chlorite (ClO2 - ) by the three nitrate reductases (NarA, NarZ, and Nap) led to methionine oxidation of periplasmic proteins. In response to this stress, the E. coli HprSR two-component system was activated, leading to the over-production of MsrPQ. This study, therefore, supports the idea that methionine oxidation in proteins is part of chlorate toxicity, and that MsrPQ can be considered as an anti-chlorate/chlorite defense system in bacteria. Finally, this study challenges the traditional view of the absence of Met-oxidation during anaerobiosis.

Refolding and biophysical characterization of the Caulobacter crescentus copper resistance protein, PcoB: An outer membrane protein containing an intrinsically disordered domain.

Copper cations play fundamental roles in biological systems, such as protein folding and stabilization, or enzymatic reactions. Although copper is essential to the cell, it can become cytotoxic if present in too high concentration. Organisms have therefore developed specific regulation mechanisms towards copper. This is the case of the Pco system present in the bacterium Caulobacter crescentus, which is composed of two proteins: a soluble periplasmic protein PcoA and an outer membrane protein PcoB. PcoA oxidizes Cu+ to Cu2+, whereas PcoB is thought to be an efflux pump for Cu2+. While the PcoA protein has already been studied, very little is known about the structure and function of PcoB. In the present work, PcoB has been overexpressed in high yield in E. coli strains and successfully refolded by the SDS-cosolvent method. Binding to divalent cations has also been studied using several spectroscopic techniques. In addition, a three-dimensional structure model of PcoB, experimentally supported by circular dichroism, has been constructed, showing a ß-barrel conformation with a N-terminal disordered chain. This peculiar intrinsic disorder property has also been confirmed by various bioinformatic tools.

Periplasmic Bacterial Biomineralization of Copper Sulfide Nanoparticles.

Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3 S4 ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe3 S4 ) and magnetite (Fe3 O4 ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

New insights into the mechanism of iron transport through the bacterial Ftr system present in pathogens.

Iron is an essential nutrient in bacteria. Its ferrous form, mostly present in low oxygen and acidic pH environments, can be imported using the specific Ftr-type transport system, which encompasses the conserved FtrABCD system found in pathogenic bacteria such as Bordetella, Brucella and Burkholderia. The nonpathogenicity and versatile metabolism of Rubrivivax gelatinosus make it an ideal model to study the FtrABCD system. Here, we report a new aspect of its regulation and the role of the periplasmic proteins FtrA and FtrB using in vivo and in vitro approaches. We investigated the metal binding mode and redox state of copper and iron to FtrA by crystallography and biophysical methods. An 'as isolated' FtrA protein from the bacterial periplasm contained a copper ion (Cu+ ) identified by electron paramagnetic resonance (EPR). Copper is coordinated by four conserved side chains (His and Met) in the primary metal site. Structural analysis of R. gelatinosus FtrA and FtrA homologues revealed that copper binding induces a rearrangement of the His95 imidazole ring, releasing thereafter space, as well as both Asp45 and Asp92 side chains, for iron binding in the secondary metal site. EPR highlighted that FtrA can oxidize the bound ferrous ion into the ferric form by reducing the bound Cu2+ into Cu+ , both metal sites being separated by 7 Å. Finally, we showed that FtrB binds iron and not copper. These results provide new insights into the mechanism of ferrous iron utilization by the conserved FtrABCD iron transporter for which we propose a new functional model.

Insights into the client protein release mechanism of the ATP-independent chaperone Spy.

Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.

Synergy of protease-binding sites within the ecotin homodimer is crucial for inhibition of MASP enzymes and for blocking lectin pathway activation.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Structural basis of copper binding by a dimeric periplasmic protein forming a six-helical bundle.

Bacteria maintain copper balance by various copper response mechanisms. A plasmid gene encoding a methionine rich protein targeted to periplasm is adjacent to the sil operon that confers heavy metal resistance. However, the gene product Orf91 has not been characterized before. Using X-ray crystallography, we solved the structures of Orf91 in apo, cuprous ion-bound, and cupric ion-bound forms. An Orf91 protomer consists of three helices of which the C-terminal two helices belong to domain of unknown function 305 (DUF305), and two Orf91s dimerize into a six-helical bundle. The MxxHH motif specific for DUF305 is critical for cuprous ion binding, and the MxxMxxMHxxMM motif in the N-terminal helix contributes to cupric ion binding. The first histidine of MxxHH shows alternative conformations related to the redox state of copper ion. We suggest that Orf91 is an adaptable copper sponge in the periplasmic space.