Human calpain-3 and its structural plasticity: dissociation of a homohexamer into dimers on binding titin.

Calpain-3 is an intracellular Ca2+-dependent cysteine protease abundant in skeletal muscle. Its physiological role in the sarcomere is thought to include removing damaged muscle proteins after exercise. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These mutations, of which there are over 500 in humans, are spread all along this 94-kDa multi-domain protein that includes three 40+-residue sequences (NS, IS1, and IS2). The latter sequences are unique to this calpain isoform and are hypersensitive to proteolysis. To investigate the whole enzyme structure and how mutations might affect its activity, we produce the proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in E. coli. During size-exclusion chromatography, this calpain-3 was consistently eluted as a much larger 0.5-MDa complex rather than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS, Blue Native PAGE, and AUC, made the complex amenable to single-particle cryo-EM analysis. From two data sets, we obtained a 3.85-Å reconstruction map that shows the complex is a trimer of calpain-3 dimers with six penta-EF-hand domains at its core. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When this 37-kDa region of titin was co-expressed with calpain-3 the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to several copies of the titin fragment. We suggest that newly synthesized calpain-3 is kept as an inactive hexamer until it binds the N2A region of titin in the sarcomere, whereupon it dissociates into functional dimers.

Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin.

Analogs of proline can be used to expand the chemical space about the residue while maintaining its uniquely restricted conformational space. Here, we demonstrate the incorporation of 4R-methylproline, 4S-methylproline, and 4-methyleneproline into recombinant insulin expressed in Escherichia coli. These modified proline residues, introduced at position B28, change the biophysical properties of insulin: Incorporation of 4-methyleneproline at B28 accelerates fibril formation, while 4-methylation speeds dissociation from the pharmaceutically formulated hexamer. This work expands the scope of proline analogs amenable to incorporation into recombinant proteins and demonstrates how noncanonical amino acid mutagenesis can be used to engineer the therapeutically relevant properties of protein drugs.

How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization.

The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila PhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker.

Characterization and quantification of adeno-associated virus capsid-loading states by multi-wavelength analytical ultracentrifugation with UltraScan.

Aim: We present multi-wavelength (MW) analytical ultracentrifugation (AUC) methods offering superior accuracy for adeno-associated virus characterization and quantification. Methods: Experimental design guidelines are presented for MW sedimentation velocity and analytical buoyant density equilibrium AUC. Results: Our results were compared with dual-wavelength AUC, transmission electron microscopy and mass photometry. In contrast to dual-wavelength AUC, MW-AUC correctly quantifies adeno-associated virus capsid ratios and identifies contaminants. In contrast to transmission electron microscopy, partially filled capsids can also be detected and quantified. In contrast to mass photometry, first-principle results are obtained. Conclusion: Our study demonstrates the improved information provided by MW-AUC, highlighting the utility of several recently integrated UltraScan programs, and reinforces AUC as the gold-standard analysis for viral vectors.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

A new UltraScan module for the characterization and quantification of analytical buoyant density equilibrium experiments to determine AAV capsid loading.

A method for characterizing and quantifying peaks formed in an analytical buoyant density equilibrium (ABDE) experiment is presented. An algorithm is derived to calculate the concentration of the density forming gradient material at every point in the cell, provided the rotor speed, temperature, meniscus position, bottom of the cell position, and the loading concentration, molar mass, and partial specific volume of the density gradient-forming material are known. In addition, a new peak fitting algorithm has been developed which allows the user to automatically quantify the peaks formed in terms of density, apparent partial specific volume, and relative abundance. The method is suitable for both ionic and non-ionic density forming materials and can be used with data generated from the UV optical system as well as the AVIV fluorescence optical system. These methods have been programmed in a new UltraScan-III module (us_abde). Examples are shown that demonstrate the application of the new module to adeno-associated viral vector preparations and proteins.

Investigating RNA-RNA interactions through computational and biophysical analysis.

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5' and 3' terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

Hollow Octadecameric Self-Assembly of Collagen-like Peptides.

The folding of collagen is a hierarchical process that starts with three peptides associating into the characteristic triple helical fold. Depending on the specific collagen in question, these triple helices then assemble into bundles reminiscent of α-helical coiled-coils. Unlike α-helices, however, the bundling of collagen triple helices is very poorly understood with almost no direct experimental data available. In order to shed light on this critical step of collagen hierarchical assembly, we have examined the collagenous region of complement component 1q. Thirteen synthetic peptides were prepared to dissect the critical regions allowing for its octadecameric self-assembly. We find that short peptides (under 40 amino acids) are able to self-assemble into specific (ABC)6 octadecamers. This requires the ABC heterotrimeric composition as the self-assembly subunit, but does not require disulfide bonds. Self-assembly into this octadecamer is aided by short noncollagenous sequences at the N-terminus, although they are not entirely required. The mechanism of self-assembly appears to begin with the very slow formation of the ABC heterotrimeric helix, followed by rapid bundling of triple helices into progressively larger oligomers, terminating in the formation of the (ABC)6 octadecamer. Cryo-electron microscopy reveals the (ABC)6 assembly as a remarkable, hollow, crown-like structure with an open channel approximately 18 Å at the narrow end and 30 Å at the wide end. This work helps to illuminate the structure and assembly mechanism of a critical protein in the innate immune system and lays the groundwork for the de novo design of higher order collagen mimetic peptide assemblies.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria.

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription.

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats.

Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions.

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism.

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations.

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor's regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135-555) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135-555, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135-555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135-555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135-555 interacting with and unwinding RVFV non-coding regions.