Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Nanobody Loop Mimetics Enhance Son of Sevenless 1-Catalyzed Nucleotide Exchange on RAS.

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active guanosine triphosphate (GTP)-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14 (Nb14), which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of Nb14 with an EC50 value of 29 µM. Altogether, this study demonstrated that peptides able to modulate a protein-protein interaction can be obtained by structural mimicry of a Nb paratope.

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2.

Inhibition of transcriptional regulators of bacterial pathogens with the aim of reprogramming their metabolism to modify their antibiotic susceptibility constitutes a promising therapeutic strategy. One example is the bio-activation of the anti-tubercular pro-drug ethionamide, which activity could be enhanced by inhibiting the transcriptional repressor EthR. Recently, we discovered that inhibition of a second transcriptional repressor, EthR2, leads to the awakening of a new ethionamide bio-activation pathway. The x-ray structure of EthR2 was solved at 2.3 Å resolution in complex with a compound called SMARt-420 (Small Molecule Aborting Resistance). Detailed comparison and structural analysis revealed interesting insights for the upcoming structure-based design of EthR2 inhibitors as an alternative to revert ethionamide resistance in Mycobacterium tuberculosis.

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

BACKGROUND: The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. RESULTS: To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. CONCLUSIONS: Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Probing the N-terminal ß-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP(C), into its misfolded oligomeric form, known as prion or PrP(Sc). The canonical human PrP(C) (HuPrP) fold features an unstructured N-terminal part (residues 23-124) and a well-defined C-terminal globular domain (residues 125-231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113-120) plays an important role in the conversion to PrP(Sc). The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel ß-sheet with the ß1 and ß2 strands, demonstrating that the full-length HuPrP(C) can adopt a more elaborate ß0-ß1-α1-ß2-α2-α3 structural organization than the canonical ß1-α1-ß2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates ß-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP(C) into PrP(Sc).

The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb.

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

Allosteric nanobodies to study the interactions between SOS1 and RAS.

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.

Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.

High-resolution structure of a papaya plant-defense barwin-like protein solved by in-house sulfur-SAD phasing.

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Šwavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central ß-barrel of six strands surrounded by several α-helices and loops. The ß-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

Characterization of the PAS domain in the sensor-kinase BvgS: mechanical role in signal transmission.

BACKGROUND: In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37°C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap domains and a cytoplasmic PAS domain that precedes the kinase domain, which is followed by additional phosphotransfer domains. PAS domains are small, ubiquitous sensing or regulatory domains. The function of the PAS domain in BvgS remains unknown. RESULTS: We showed that recombinant BvgS PAS proteins form dimers that are stabilized by α helical regions flanking the PAS core. A structural model of the PAS domain dimer was built and probed by site-directed mutagenesis and by biochemical and functional analyses. Although we found no ligands for the PAS domain cavity, its integrity is required for signaling. We also showed that the structural stability of the PAS core and its proper coupling to its flanking N- and C-terminal α helices are crucial for BvgS activity. CONCLUSIONS: We propose that a major function of the BvgS PAS domain is to maintain conformational signals arising from mechanical strain generated by the periplasmic domain. The tight structure of the PAS core and its connections with the upstream and downstream helices ensure signaling to the kinase domain, which determines BvgS activity. Many mild substitutions that map to the PAS domain keep BvgS active but make it unresponsive to negative modulators, supporting that modulation increases conformational strain in the protein.

The mannose-specific lectin domains of Flo1p from Saccharomyces cerevisiae and Lg-Flo1p from S. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin-carbohydrate complexes.

Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

A novel expression system for production of soluble prion proteins in E. coli.

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding ß-lactamase in complex with its ligand.

TEM-1 ß-lactamase is a highly efficient enzyme that is involved in bacterial resistance against ß-lactam antibiotics such as penicillin. It is also a robust scaffold protein which can be engineered by molecular-evolution techniques to bind a variety of targets. One such ß-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native ß-lactamases. In addition to recognizing kan, BlaKr activity is up-regulated by its binding via an activation mechanism which is not yet understood at the molecular level. In order to fill this gap, determination of the structure of the BlaKr-kan complex was embarked upon. A crystallization condition for BlaKr-kan was identified using high-throughput screening, and crystal growth was further optimized using streak-seeding and hanging-drop methods. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.01, b = 72.33, c = 74.62 Å, and diffracted to 1.67 Šresolution using synchrotron radiation. The X-ray structure of BlaKr with its ligand kanamycin should provide the molecular-level details necessary for understanding the activation mechanism of the engineered enzyme.

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein.

Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=30.04, b=37.15, c=83.00 Å, and diffracted to 1.23 Šresolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility.

Phthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachidonyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with vancomycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates.

Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity.

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection.

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.