The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the host antiviral response.

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non-structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5'triphosphorylated double-stranded RNA (5'-ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5'ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5'ppp-dsRNA, and thus suppresses activation of RIG-I's ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5'ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5'ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5'ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

Structure Determination of Challenging Protein-Peptide Complexes Combining NMR Chemical Shift Data and Molecular Dynamics Simulations.

Intrinsically disordered regions of proteins often mediate important protein-protein interactions. However, the folding-upon-binding nature of many polypeptide-protein interactions limits the ability of modeling tools to predict the three-dimensional structures of such complexes. To address this problem, we have taken a tandem approach combining NMR chemical shift data and molecular simulations to determine the structures of peptide-protein complexes. Here, we use the MELD (Modeling Employing Limited Data) technique applied to polypeptide complexes formed with the extraterminal domain (ET) of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can adopt different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully modeled bound-state conformations and binding poses, using only protein receptor backbone chemical shift data, in excellent agreement with experimentally determined structures for moderately tight (Kd ∼100 nM) binders. The hybrid MELD + NMR approach required additional peptide ligand chemical shift data for weaker (Kd ∼250 µM) peptide binding partners. AlphaFold also successfully predicts the structures of some of these peptide-protein complexes. However, whereas AlphaFold can provide qualitative peptide rankings, MELD can directly estimate relative binding affinities. The hybrid MELD + NMR approach offers a powerful new tool for structural analysis of protein-polypeptide complexes involving disorder-to-order transitions upon complex formation, which are not successfully modeled with most other complex prediction methods, providing both the 3D structures of peptide-protein complexes and their relative binding affinities.

A common binding motif in the ET domain of BRD3 forms polymorphic structural interfaces with host and viral proteins.

The extraterminal (ET) domain of BRD3 is conserved among BET proteins (BRD2, BRD3, BRD4), interacting with multiple host and viral protein-protein networks. Solution NMR structures of complexes formed between the BRD3 ET domain and either the 79-residue murine leukemia virus integrase (IN) C-terminal domain (IN329-408) or its 22-residue IN tail peptide (IN386-407) alone reveal similar intermolecular three-stranded ß-sheet formations. 15N relaxation studies reveal a 10-residue linker region (IN379-388) tethering the SH3 domain (IN329-378) to the ET-binding motif (IN389-405):ET complex. This linker has restricted flexibility, affecting its potential range of orientations in the IN:nucleosome complex. The complex of the ET-binding peptide of the host NSD3 protein (NSD3148-184) and the BRD3 ET domain includes a similar three-stranded ß-sheet interaction, but the orientation of the ß hairpin is flipped compared with the two IN:ET complexes. These studies expand our understanding of molecular recognition polymorphism in complexes of ET-binding motifs with viral and host proteins.

A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus.

Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine.

A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145 kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1H-13C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.

A Second RNA-Binding Site in the NS1 Protein of Influenza B Virus.

Influenza viruses cause a highly contagious respiratory disease in humans. The NS1 proteins of influenza A and B viruses (NS1A and NS1B proteins, respectively) are composed of two domains, a dimeric N-terminal domain and a C-terminal domain, connected by a flexible polypeptide linker. Here we report the 2.0-Å X-ray crystal structure and nuclear magnetic resonance studies of the NS1B C-terminal domain, which reveal a novel and unexpected basic RNA-binding site that is not present in the NS1A protein. We demonstrate that single-site alanine replacements of basic residues in this site lead to reduced RNA-binding activity, and that recombinant influenza B viruses expressing these mutant NS1B proteins are severely attenuated in replication. This novel RNA-binding site of NS1B is required for optimal influenza B virus replication. Most importantly, this study reveals an unexpected RNA-binding function in the C-terminal domain of NS1B, a novel function that distinguishes influenza B viruses from influenza A viruses.

(19)F NMR reveals multiple conformations at the dimer interface of the nonstructural protein 1 effector domain from influenza A virus.

Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply (19)F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan, and we demonstrate that the (19)F signal of Trp187 is a sensitive, direct monitor of the ED helix:helix dimer interface. (19)F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein:protein interface, whose rate is more than three orders of magnitude faster than the kinetics of ED dimerization. (19)F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection.

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded ß barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.

Solution NMR structure of the ribosomal protein RP-L35Ae from Pyrococcus furiosus.

The ribosome consists of small and large subunits each composed of dozens of proteins and RNA molecules. However, the functions of many of the individual protomers within the ribosome are still unknown. In this article, we describe the solution NMR structure of the ribosomal protein RP-L35Ae from the archaeon Pyrococcus furiosus. RP-L35Ae is buried within the large subunit of the ribosome and belongs to Pfam protein domain family PF01247, which is highly conserved in eukaryotes, present in a few archaeal genomes, but absent in bacteria. The protein adopts a six-stranded anti-parallel ß-barrel analogous to the "tRNA binding motif" fold. The structure of the P. furiosus RP-L35Ae presented in this article constitutes the first structural representative from this protein domain family.

Identification of influenza virus inhibitors targeting NS1A utilizing fluorescence polarization-based high-throughput assay.

This article describes the development of a simple and robust fluorescence polarization (FP)-based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z' score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.

Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus.

Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells.

Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.

Non-structural protein 1 from influenza A virus, NS1A, is a key multifunctional virulence factor composed of two domains: an N-terminal double-stranded RNA (dsRNA)-binding domain and a C-terminal effector domain (ED). Isolated RNA-binding and effector domains of NS1A both exist as homodimers in solution. Despite recent crystal structures of isolated ED and full-length NS1A proteins from different influenza virus strains, controversy remains over the actual biologically relevant ED dimer interface. Here, we report the biophysical properties of the NS1A ED from H3N2 influenza A/Udorn/307/1972 (Ud) virus in solution. Several lines of evidence, including (15)N NMR relaxation, NMR chemical shift perturbations, static light scattering, and analytical sedimentation equilibrium, demonstrate that Ud NS1A ED forms a relatively weak dimer in solution (K(d) = 90 ± 2 µm), featuring a symmetric helix-helix dimer interface. Mutations within and near this interface completely abolish dimerization, whereas mutations consistent with other proposed ED dimer interfaces have no effect on dimer formation. In addition, the critical Trp-187 residue in this interface serves as a sensitive NMR spectroscopic marker for the concentration-dependent dimerization of NS1A ED in solution. Finally, dynamic light scattering and gel shift binding experiments demonstrate that the ED interface plays a role in both the oligomerization and the dsRNA binding properties of the full-length NS1A protein. In particular, mutation of the critical tryptophan in the ED interface substantially reduces the propensity of full-length NS1A from different strains to oligomerize and results in a reduction in dsRNA binding affinity for full-length NS1A.

Backbone and Ile-δ1, Leu, Val methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein.

Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone (1)H(N), (15)N, (13)C', and (13)C(α), as well as side chain (13)C(ß), methyl (Ile-δ1, Leu, Val), amide (Asn, Gln), and indole N-H (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.

Synthesis and evaluation of quinoxaline derivatives as potential influenza NS1A protein inhibitors.

A library of quinoxaline derivatives were prepared to target non-structural protein 1 of influenza A (NS1A) as a means to develop anti-influenza drug leads. An in vitro fluorescence polarization assay demonstrated that these compounds disrupted the dsRNA-NS1A interaction to varying extents. Changes of substituent at positions 2, 3 and 6 on the quinoxaline ring led to variance in responses. The most active compounds (35 and 44) had IC(50) values in the range of low micromolar concentration without exhibiting significant dsRNA intercalation. Compound 44 was able to inhibit influenza A/Udorn/72 virus growth.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

Structures of influenza A proteins and insights into antiviral drug targets.

The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the molecular biology of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance our understanding of the molecular mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the nonstructural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry.

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three-dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of (1)H/(2)H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N- and C-terminal tails were evaluated using (1)H-(15)N HSQC and (1)H-(15)N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS-based truncated construct for a 77-residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination.

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study.

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.