Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

Structural and dynamic characterization of the C-terminal tail of ErbB2: Disordered but not random.

ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers and associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last 20 years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, the ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics, and detailed functional mechanism. Yet, it is where signal transduction is triggered via phosphorylation of tyrosine residues and carried out via interaction with adaptor proteins. Here, we report the first description, to our knowledge, of the ErbB2 disordered tail at atomic resolution using NMR, complemented by small-angle x-ray scattering. We show that although no part of CtErbB2 has any fully populated secondary or tertiary structure, it contains several transient α-helices and numerous transient polyproline II helices, populated up to 20 and 40%, respectively, and low but significant compaction. The presence of some structural elements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2's autoregulation processes. In addition, the transient formation of polyproline II helices is compliant with previously suggested interactions with SH3 domains. All in all, our in-depth structural study opens perspectives in the mechanistic understanding of ErbB2.

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners.

Phosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that N- and C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase.

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

UNLABELLED: Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCE: Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs.

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.

Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.

Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177) core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177), as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies.

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design.

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

Epitope convergence of broadly HIV-1 neutralizing IgA and IgG antibody lineages in a viremic controller.

Decrypting the B cell ontogeny of HIV-1 broadly neutralizing antibodies (bNAbs) is paramount for vaccine design. Here, we characterized IgA and IgG bNAbs of three distinct B cell lineages in a viremic controller, two of which comprised only IgG+ or IgA+ blood memory B cells; the third combined both IgG and IgA clonal variants. 7-269 bNAb in the IgA-only lineage displayed the highest neutralizing capacity despite limited somatic mutation, and delayed viral rebound in humanized mice. bNAbs in all three lineages targeted the N332 glycan supersite. The 2.8-Å resolution cryo-EM structure of 7-269-BG505 SOSIP.664 complex showed a similar pose as 2G12, on an epitope mainly composed of sugar residues comprising the N332 and N295 glycans. Binding and cryo-EM structural analyses showed that antibodies from the two other lineages interact mostly with glycans N332 and N386. Hence, multiple B cell lineages of IgG and IgA bNAbs focused on a unique HIV-1 site of vulnerability can codevelop in HIV-1 viremic controllers.

NMR structure of the human Mediator MED25 ACID domain.

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed ß-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the ß-barrel, defined by strands B4-B7-B6.

Probing the relationship between Gram-negative and Gram-positive S1 proteins by sequence analysis.

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine-Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed 'S1 proteins'. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their beta-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4-5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1.

A Structural and Dynamic Analysis of the Partially Disordered Polymerase-Binding Domain in RSV Phosphoprotein.

The phosphoprotein P of Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD-PCα region in solution, at the single residue level by NMR and at lower resolution by complementary biophysical methods. We probed POD-PCα inter-domain contacts and showed that small molecules were able to modify the dynamics of PCα. These structural properties are fundamental to the peculiar binding mode of RSV phosphoprotein to L, where each of the four protomers binds to L in a different way.

The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases.

BACKGROUND: Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. RESULTS: We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. CONCLUSIONS: The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

Activation of RegB endoribonuclease by S1 ribosomal protein requires an 11 nt conserved sequence.

The T4 RegB endoribonuclease cleaves specifically in the middle of the -GGAG- sequence, leading to inactivation and degradation of early phage mRNAs. In vitro, RegB activity is very weak but can be enhanced 10- to 100-fold by the Escherichia coli ribosomal protein S1. Not all RNAs carrying the GGAG motif are cleaved by RegB, suggesting that additional information is required to obtain a complete RegB target site. In this work, we find that in the presence of S1, the RegB target site is an 11 nt long single-stranded RNA carrying the 100% conserved GGA triplet at the 5' end and a degenerate, A-rich, consensus sequence immediately downstream. Our data support the notion that RegB alone recognizes only the trinucleotide GGA, which it cleaves very inefficiently, and that stimulation of RegB activity by S1 depends on the nucleotide immediately 3' to -GGA-.

A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar coupling and small angle X-ray scattering data.

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the gammaS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active.

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using 31P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2',3'-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by 1H/15N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.

Mutations in actin used for structural studies partially disrupt ß-thymosin/WH2 domains interaction.

Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model ß-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both ß-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control.

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.

Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

Ribosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E. coli S1 is a modular protein that contains six repeats of an S1 motif, which have distinct functions despite structural homology. Whereas the three central repeats have been shown to be involved in mRNA recognition, the two first repeats that constitute the N-terminal domain of S1 are responsible for binding to the 30S subunit. Here we report the almost complete (1)H, (13)C and (15)N resonance assignment of two fragments of the 30S binding region of S1. The first fragment comprises only the first repeat. The second corresponds to the entire ribosome binding domain. Since S1 is absent from all high resolution X-ray structures of prokaryotic ribosomes, these data provide a first step towards atomic level structural characterization of this domain by NMR. Chemical shift analysis of the first repeat provides evidence for structural divergence from the canonical OB-fold of an S1 motif. In contrast the second domain displays the expected topology for an S1 motif, which rationalizes the functional specialization of the two subdomains.