Β-barrel Proteins Research Articles

Optimizing the function and dynamics of de novo designed fluorescent β-barrel proteins.

Protein design originally began as a field intended to demonstrate our understanding of how proteins fold. Over the last three decades, advances in design principles and algorithms have paved the way for de novo computational design of new classes of proteins. While biological or drug-like applications for these proteins have been demonstrated, existing computational techniques are generally not able to predict whether a design will be highly or only moderately functional. We have used computational simulation and modelling to study the mini-fluorescence activating-protein (mFAP) that was designed to function as a fluorescent marker or biosensor when bound to an exogenous chromophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). DFHBI fluoresces when it is in a planar-Z conformation and mFAPs stabilize DFHBI in this configuration, increasing fluorescence 10-100x fold relative to free DFHBI in solution. However, when compared to eGFP they are about 5-fold less efficient (i.e., dimmer). In our latest work, we have built a predictive model that accurately captures the photophysical behavior DFHBI in the binding pocket of 13 mFAP designs via molecular dynamics (MD) simulations. When analyzing the brightest mFAP variant, we have found that certain side-chain rotational isomers (rotamers) are strongly associated with keeping DFHBI in a planar-Z conformation and thus a preference for conformationally shifting towards functional states. Because of the high computational cost for running MD simulations, we are in the process of developing a high-throughput, low-cost algorithm—using the recently developed MDR-FEP method—to find mutants that increase the existence of these rotamers.

A multipoint guidance mechanism for β-barrel folding on the SAM complex.

Mitochondrial β-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic β-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last β-strand (β-signal) of the substrate-the 19-β-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first β-segment (β1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces β1 to promote its pairing with Tom40's last β-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial β-barrel folding.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane β-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

Repurposing a Nitric Oxide Transport Hemoprotein Nitrophorin 2 for Olefin Cyclopropanation

A growing number of heme proteins have recently been repurposed for catalyzing abiological carbene transfer reactions. Herein, we rationally designed an engineered variant of nitrophorin 2 (NP2)─a nitric oxide transport hemoprotein─that catalyzes olefin cyclopropanation with high activity and stereoselectivity. Being a β-barrel protein, the engineered NP2 variant showed a unique substrate preference, in contrast to the mainstream α-helical carbene-transfer heme enzymes like cytochrome P450 enzymes and myoglobin. The catalytic reactions can be carried out on a preparative scale while maintaining the stereoselectivity. The stereoselectivity of the NP2-catalyzed styrene cyclopropanation was further supported by quantum chemical calculations, and the significance of key residues was elucidated. As such, this work establishes NP2 as a robust lipocalin scaffold amenable for carbene-transferase development, complementing the current biocatalytic toolbox.

Design and optimization of enzymatic activity in a de novo β-barrel scaffold.

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β‐barrel protein to create catalysts for a retro‐aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Challenges for the development of a universal vaccine against leptospirosis revealed by the evaluation of 22 vaccine candidates.

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane β-barrel proteins (βb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral βb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral βb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more βb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 – 125 μg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 – 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 – 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

An Unprecedented Tolerance to Deletion of the Periplasmic Chaperones SurA, Skp, and DegP in the Nosocomial Pathogen Acinetobacter baumannii.

ABSTRACTThe outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants, or dryness. The main constituents of the OM are integral OM β-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa, the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage; the chaperones SurA, Skp, and DegP, which facilitate the passage of β-barrel OMPs through the periplasm; and the β-barrel assembly machinery (BAM), which facilitates insertion into the OM. In E. coli, Y. enterocolitica, and P. aeruginosa, the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotic treatment, and reduced virulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii, we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to E. coli, Y. enterocolitica, and P. aeruginosa, AB5075 tolerates the lack of SurA, Skp, or DegP with only weak mutant phenotypes. While the double knockout strains ΔsurAΔskp and ΔsurAΔdegP are conditionally lethal in E. coli, all double deletions were well tolerated by AB5075. Strikingly, even a triple-knockout strain of AB5075, lacking surA, skp, and degP, was viable.IMPORTANCE Acinetobacter baumannii is a major threat to human health due to its ability to persist in the hospital environment, resistance to antibiotic treatment, and ability to deploy multiple and redundant virulence factors. In a rising number of cases, infections with multidrug-resistant A. baumannii end up fatally, because all antibiotic treatment options fail. Thus, novel targets have to be identified and alternative therapeutics have to be developed. The knockout of periplasmic chaperones has previously proven to significantly reduce virulence and even break antibiotic resistance in other Gram-negative pathogens. Our study in A. baumannii demonstrates how variable the importance of the periplasmic chaperones SurA, Skp, and DegP can be and suggests the existence of mechanisms allowing A. baumannii to cope with the lack of the three periplasmic chaperones.

Evidence for a Widespread Third System for Bacterial Polysaccharide Export across the Outer Membrane Comprising a Composite OPX/β-Barrel Translocon.

ABSTRACTIn Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other and interact in in vivo pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning β-barrel translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria.

Slowly Relaxing Local Structure Analysis of 15N Relaxation from the Proteins p50 and Human Neutrophil Gelatinase-Associated Lipocalin: New Insights into the Dynamic Structure of β-Barrel Proteins

Nuclear magnetic resonance relaxation analysis is a powerful method for studying the internal mobility of proteins. We have developed for analysis the slowly relaxing local structure (SRLS) approach. SRLS is general in its nature in several respects, including the tensorial representation of the physical quantities comprising the dynamic model. By controlling tensor symmetry, a broad range of systems can be treated with physical relevance, typically with data-fitting techniques. In simple limits, SRLS yields the traditional model-free (MF) method. In the present context, MF simplicity means featuring the highest possible tensor symmetry. This renders MF-based data-fitting susceptible to the usage of fit parameters, yielding physically ill-defined results. A typical candidate is the Rex term, devised to represent ms-μs motions but often invoked by the fitting scheme just to improve the statistics. Here, we consider two such cases using the N-H bond as probe and the proteins p50 and human neutrophil gelatinase-associated lipocalin as paradigm systems. We illustrate the harm caused by the physically unjustified involvement of Rex in MF-based 15N relaxation analysis. Then, we show that forgoing the usage of Rex, SRLS analysis of the very same experimental data provides interesting new information.

SfGFP throws light on the early stages of β-barrel amyloidogenesis

The accumulation of β-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native β-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic β-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate “native-like” state preserving the integrity of the sfGFP β-barrel scaffold despite the partial distortion of the three β-strands closing it. The interaction between the “melted” regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the β-barrel opens and some of the native α-helices and disordered regions refold into non-native β-strands, which, along with native β-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other β-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis.

Crowding-induced protein destabilization in the absence of soft attractions

It is generally assumed that volume exclusion by macromolecular crowders universally stabilizes the native states of proteins and destabilization suggests soft attractions between crowders and protein. Here we show that proteins can be destabilized even by crowders that are purely repulsive. With a coarse-grained sequence-based model, we study the folding thermodynamics of two sequences with different native folds, a helical hairpin and a β-barrel, in a range of crowder volume fractions, φc. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with φc. Hence, for all φc>0, U is entropically disfavored relative to N. This entropy-centric view holds for the helical hairpin protein, which is stabilized under all crowded conditions as quantified by changes in either the folding midpoint temperature, Tm, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-φc conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at T<Tm, such that U is only weakly disfavored entropically by the crowders; and 2) a transient, compact, and relatively low-energy nonnative state that has a maximum population of only a few percent at φc=0, but increasing monotonically with φc. Overall, protein destabilization driven by hard-core effects appears possible when a compaction of U leads to even a modest population of compact nonnative states that are energetically competitive with N.

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of β-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

From Alpha to Beta – a co-translational way to fold?

ABSTRACT Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion.

Strategy for optimized expression of beta-barrel proteins from pathogenic Gram-negative bacteria to the outer membrane of Escherichia coli

Outer membranes (OM) of Gram-negative bacteria often contain essential components for pathogen-host interactions, infection and disease progression of pathogenic species. Such virulence factors include, but are not limited to, β-barrel outer membrane proteins and modified lipids, such as lipopolysaccharide (LPS). Investigating β-barrel outer membrane proteins in a more native environment can often be challenging in vitro due to difficulties in expression to and purification from the membrane. Studying virulence factors of pathogenic bacteria in vivo is also complicated by the need for higher biosafety lab facilities.

Comparing the dynamics of the BAM complex between two species

Outer membrane proteins (OMPs) in Gram-negative bacteria are transmembrane β-barrel proteins that are involved in important biological activities such as nutrient transport, signal transduction and the export of virulence factors. While it’s known that the protein complex responsible for assembly and insertion of OMPs into the outer membrane is the β-barrel assembly machinery (BAM), the detailed mechanisms for OMP insertion by BAM are only recently coming into focus. Nonetheless, to date, the only complete high-resolution structures of BAM are from Escherichia coli, leaving open the question of the degree of conservation of mechanisms across species.

A switch from α‐helical to β‐strand conformation during co‐translational protein folding

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo‐EM structure determination to show that folding of a β‐barrel protein begins with formation of a dynamic α‐helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N‐terminal part of the nascent chain refolds to a β‐hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α‐helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl‐transferase center suggest that protein folding could modulate ribosome activity.

Characterization of the O-Glycoproteome of Porphyromonas gingivalis.

ABSTRACTPorphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm.IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0–2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.

Modulation of host cellular responses by gram-negative bacterial porins.

The outer membrane of a gram-negative bacteria encapsulates the plasma membrane thereby protecting it from the harsh external environment. This membrane acts as a sieving barrier due to the presence of special membrane-spanning proteins called "porins." These porins are β-barrel channel proteins that allow the passive transport of hydrophilic molecules and are impermeable to large and charged molecules. Many porins form trimers in the outer membrane. They are abundantly present on the bacterial surface and therefore play various significant roles in the host-bacteria interactions. These include the roles of porins in the adhesion and virulence mechanisms necessary for the pathogenesis, along with providing resistance to the bacteria against the antimicrobial substances. They also act as the receptors for phage and complement proteins and are involved in modulating the host cellular responses. In addition, the potential use of porins as adjuvants, vaccine candidates, therapeutic targets, and biomarkers is now being exploited. In this review, we focus briefly on the structure of the porins along with their important functions and roles in the host-bacteria interactions.