In Vivo Protein-Protein Binding Competition Assay Based on Split-GFP Reassembly: Proof of Concept.

The split-green fluorescent protein (GFP) reassembly assay is a well-established approach to study protein-protein interactions (PPIs). In this assay, when two interacting proteins X and Y, respectively fused to residues 1-157 and to residues 158-237 of GFP, are co-expressed in E. coli, the two GFP halves are brought to sufficient proximity to reassociate and fold to recreate the functional GFP. At constant protein expression level, the intensity of fluorescence produced by the bacteria is proportional to the binding affinity of X to Y. We hypothesized that adding a third partner (Z) endowed with an affinity for either X or Y would lead to an in vivo competition assay. We report here the different steps of the set-up of this competition assay, and define the experimental conditions required to obtained reliable results. Results show that this competition assay is a potentially interesting tool for screening libraries of binding inhibitors, Z being either a protein or a chemical reagent.

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis.

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four ß-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results.

Functional benefit of structural disorder for the replication of measles, Nipah and Hendra viruses.

Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid-liquid phase separation and fibrillation.

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins.

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a ß-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200-310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

Structural and Functional Characterization of the ABA-Water Deficit Stress Domain from Wheat and Barley: An Intrinsically Disordered Domain behind the Versatile Functions of the Plant Abscissic Acid, Stress and Ripening Protein Family.

The ASR protein family has been discovered thirty years ago in many plant species and is involved in the tolerance of various abiotic stresses such as dehydration, salinity and heat. Despite its importance, nothing is known about the conserved ABA-Water Deficit Stress Domain (ABA-WDS) of the ASR gene family. In this study, we characterized two ABA-WDS domains, isolated from durum wheat (TtABA-WDS) and barley (HvABA-WDS). Bioinformatics analysis shows that they are both consistently predicted to be intrinsically disordered. Hydrodynamic and circular dichroism analysis indicate that both domains are largely disordered but belong to different structural classes, with HvABA-WDS and TtABA-WDS adopting a PreMolten Globule-like (PMG-like) and a Random Coil-like (RC-like) conformation, respectively. In the presence of the secondary structure stabilizer trifluoroethanol (TFE) or of increasing glycerol concentrations, which mimics dehydration, the two domains acquire an α-helical structure. Interestingly, both domains are able to prevent heat- and dehydration-induced inactivation of the enzyme lactate dehydrogenase (LDH). Furthermore, heterologous expression of TtABA-WDS and HvABA-WDS in the yeast Saccharomyces cerevisiae improves its tolerance to salt, heat and cold stresses. Taken together our results converge to show that the ABA-WDS domain is an intrinsically disordered functional domain whose conformational plasticity could be instrumental to support the versatile functions attributed to the ASR family, including its role in abiotic stress tolerance. Finally, and after validation in the plant system, this domain could be used to improve crop tolerance to abiotic stresses.

Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

Binding induced folding: Lessons from the kinetics of interaction between NTAIL and XD.

Intrinsically Disordered Proteins (IDPs) are a class of protein that exert their function despite lacking a well-defined three-dimensional structure, which is sometimes achieved only upon binding to their natural ligands. This feature implies the folding of IDPs to be generally coupled with a binding event, representing an interesting challenge for kinetic studies. In this review, we recapitulate some of the most important findings of IDPs binding-induced folding mechanisms obtained by analyzing their binding kinetics. Furthermore, by focusing on the interaction between the Measles virus NTAIL protein, a prototypical IDP, and its physiological partner, the X domain, we recapitulate the major theoretical and experimental approaches that were used to describe binding induced folding.

An arsenal of methods for the experimental characterization of intrinsically disordered proteins - How to choose and combine them?

In this review, we detail the most common experimental approaches to assess and characterize protein intrinsic structural disorder, with the notable exception of NMR and EPR spectroscopy, two ideally suited approaches that will be described in depth in two other reviews within this special issue. We discuss the advantages, the limitations, as well as the caveats of the various methods. We also describe less common and more demanding approaches that enable achieving further insights into the conformational properties of IDPs. Finally, we present recent developments that have enabled assessment of structural disorder in living cells, and discuss the currently available methods to model IDPs as conformational ensembles.

Understanding Intramolecular Crosstalk in an Intrinsically Disordered Protein.

The interaction between NTAIL and XD from the measles virus represents a paradigmatic example of molecular recognition between an intrinsically disordered protein and a folded partner. By binding to XD, a small portion of NTAIL (classically denoted as MoRE) undergoes a disorder-to-order transition, populating an α-helical structure, while the reminder of the protein remains disordered. Here, we demonstrate an unexpected crosstalk between such a disordered region and the adjacent molecular recognition element (MoRE). This result was obtained by producing a series of truncation and site-directed variants of NTAIL while measuring the effects on the kinetics of folding and binding. We show that the disordered region of NTAIL exerts its inhibitory role by slowing the folding step of the MoRE, thereby tuning the affinity of the interaction.

Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites.

In this paper we review our recent findings on the different interaction mechanisms of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL, a model viral intrinsically disordered protein (IDP), with two of its known binding partners, i.e., the C-terminal X domain of the phosphoprotein of MeV XD (a globular viral protein) and the heat-shock protein 70 hsp70 (a globular cellular protein). The NTAIL binds both XD and hsp70 via a molecular recognition element (MoRE) that is flanked by two fuzzy regions. The long (85 residues) N-terminal fuzzy region is a natural dampener of the interaction with both XD and hsp70. In the case of binding to XD, the N-terminal fuzzy appendage of NTAIL reduces the rate of α-helical folding of the MoRE. The dampening effect of the fuzzy appendage on XD and hsp70 binding depends on the length and fuzziness of the N-terminal region. Despite this similarity, NTAIL binding to XD and hsp70 appears to rely on completely different requirements. Almost any mutation within the MoRE decreases XD binding, whereas many of them increase the binding to hsp70. In addition, XD binding is very sensitive to the α-helical state of the MoRE, whereas hsp70 is not. Thus, contrary to hsp70, XD binding appears to be strictly dependent on the wild-type primary and secondary structure of the MoRE.

Experimental Characterization of Fuzzy Protein Assemblies: Interactions of Paramyxoviral NTAIL Domains With Their Functional Partners.

In this chapter we detail various experimental approaches to characterize the fuzziness of complexes made of the C-terminal domain of the nucleoprotein (NTAIL) from three representative paramyxoviruses and of the C-terminal X domain (XD) of the homologous phosphoprotein. We discuss the advantages, the limitations, as well as the caveats of the various methods. We describe experimental data showing that paramyxoviral NTAIL-XD complexes are characterized by a considerable amount of conformational heterogeneity. We also detail recent data that revealed that NTAIL is highly malleable, i.e., it displays a partner-mediated polymorphism. All the results suggest that NTAIL plasticity and fuzziness play a role in the coordination and regulation of the NTAIL interaction network so as to ensure efficient transcription and replication.

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (NTAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, NTAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the NTAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both NTAIL/XD and NTAIL/hsp70 interactions. Although the same NTAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased NTAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.

InSiDDe: A Server for Designing Artificial Disordered Proteins.

InSiDDe (In Silico Disorder Design) is a program for the in silico design of intrinsically disordered proteins of desired length and disorder probability. The latter is assessed using IUPred and spans values ranging from 0.55 to 0.95 with 0.05 increments. One to ten artificial sequences per query, each made of 50 to 200 residues, can be generated by InSiDDe. We describe the rationale used to set up InSiDDe and show that an artificial sequence of 100 residues with an IUPred score of 0.6 designed by InSiDDe could be recombinantly expressed in E. coli at high levels without degradation when fused to a natural molecular recognition element (MoRE). In addition, the artificial fusion protein exhibited the expected behavior in terms of binding modulation of the specific partner recognized by the MoRE. To the best of our knowledge, InSiDDe is the first publicly available software for the design of intrinsically disordered protein (IDP) sequences. InSiDDE is publicly available online.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions.

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (NTAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of NTAIL remains conspicuously disordered, with α-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on NTAIL /XD binding, we generated N-terminal truncation variants of NTAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV NTAIL binding partner), and for the homologous NTAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV NTAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region.

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

Order and Disorder in the Replicative Complex of Paramyxoviruses.

In this review we summarize available data showing the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely the measles (MeV), Nipah (NiV) and Hendra (HeV) viruses. We provide a detailed description of the molecular mechanisms that govern the disorder-to-order transition that the intrinsically disordered C-terminal domain (NTAIL) of their N proteins undergoes upon binding to the C-terminal X domain (XD) of the homologous P proteins. We also show that a significant flexibility persists within NTAIL-XD complexes, which therefore provide illustrative examples of "fuzziness". The functional implications of structural disorder for viral transcription and replication are discussed in light of the ability of disordered regions to establish a complex molecular partnership and to confer a considerable reach to the elements of the replicative machinery.

Improving the soluble expression of recombinant proteins by randomly shuffling 5' and 3' coding-sequence ends.

Many structural genomics (SG) programmes rely on the design of soluble protein domains. The production and screening of large libraries to experimentally select these soluble protein-encoding constructs are limited by the technologies and efforts that can be devoted to a single target. Using basic technologies available in any laboratory, a method named `boundary shuffling' was devised to generate orientated libraries for soluble domain selection without impeding the target flow.