A Flexible and Original Architecture of Two Unrelated Zinc Fingers Underlies the Role of the Multitask P1 in RYMV Spread.

Viruses of the sobemovirus genus are plant viruses, most of which generate very important agricultural and financial losses. Among them, the rice yellow mottle virus (RYMV) is one of the most damaging pathogens devastating rice fields in Africa. RYMV infectivity and propagation rely on its protein P1, identified as a key movement and potential long-distance RNA silencing suppressor. Here we describe P1's complete 3D structure and dynamics obtained by an integrative approach combining X-Ray crystallography and NMR spectroscopy. We show that P1 is organized in two semi-independent and topologically unrelated domains, each harboring an original zinc finger. The two domains exhibit different affinities for zinc and sensitivities to oxidoreduction conditions, making the C-terminal P1 region a potential labile sensor of the plant redox status. An additional level of regulation resides on the capacity of P1 to oligomerize through its N-terminal domain. Coupling P1 structure information with site-directed mutagenesis and plant functional assays, we identified key residues in each zinc domain essential for infectivity and spread in rice tissues. Altogether, our results provide the first complete structure of a sobemoviral P1 movement protein and highlight structural and dynamical properties that may serve RYMV functions to infect and invade its host plant.

Molecular insights into the biased signaling mechanism of the µ-opioid receptor.

GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the ß-arrestin pathways through the µ-opioid receptor (µOR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific µOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists trigger µOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair ß-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

Cryo-electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex.

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

Resolving the activation mechanism of the D99N antiterminator LicT protein.

LicT is an antiterminator protein of the BglG family whose members are key players in the control of carbohydrate catabolism in bacteria. These antiterminators are generally composed of three modules, an N-terminal RNA-binding domain (CAT) followed by two homologous regulation modules (PRD1 and PRD2) that control the RNA binding activity of the effector domain via phosphorylation on conserved histidines. Although several structures of isolated domains of BglG-like proteins have been described, no structure containing CAT and at least one PRD simultaneously has yet been reported in an active state, precluding detailed understanding of signal transduction between modules. To fulfill this gap, we recently reported the complete NMR sequence assignment of a constitutively active mutant (D99N) CAT-PRD1*, which contains the effector domain and the first regulation domain of LicT. As a follow-up, we have determined and report here the 3D solution structure of this active, dimeric LicT construct (40 kDa). The structure reveals how the mutation constrains the PRD1 regulation domain into an active conformation which is transduced to CAT via a network of negatively charged residues belonging to PRD1 dimeric interface and to the linker region. In addition, our data support a model where BglG-type antitermination regulatory modules can only adopt a single conformation compatible with the active structure of the effector domain, regardless of whether activation is mediated by mutation on the first or second PRD. The linker between the effector and regulation modules appears to function as an adaptable hinge tuning the position of the functional modules.

NMR chemical shift assignment of a constitutively active fragment of the antitermination protein LicT.

LicT belongs to an essential family of bacterial antitermination proteins which bind to nascent mRNAs in order to stimulate transcription of sugar-metabolizing operons. As most of other antitermination proteins involved in carbohydrate metabolism, LicT is composed of a N-terminal RNA-binding module (CAT) and two homologous regulatory modules (PRD1 and PRD2). The activity of the CAT effector module is controlled by antagonist phosphorylations by the phosphotransferase system on conserved histidines of the two C-terminal PRDs in response to available carbon sources. Previous studies on truncated and mutant constructs have provided partial structural insight into the mechanism of signal transduction between the N-terminal RNA-binding domain and the two regulation modules. However, no structure at atomic resolution has been ever solved that contain the RNA-binding domain and a regulation module. We report the NMR assignment of a constitutively active fragment of LicT, named D99A-CAT-PRD1 or CAT-PRD1*. This fragment is composed of the RNA-binding module and the first N-terminal regulation module which bears the mutation of Asp99 to an asparagine. It is dimeric as the native protein, with a 40 kD molecular weight. The D99N mutation is sufficient to endow this fragment with a high RNA-binding constitutive activity, in a phosphorylation-free context. The assignment reported here should set the base of future NMR investigation of signal transduction between the regulatory module and the effector module in the active state of the protein, and in the long term enable the structural study of the full length protein structure in interaction with its target RNA.

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation.

LicT belongs to an essential family of bacterial transcriptional antitermination proteins controlling the expression of sugar-metabolizing operons. When activated, they bind to nascent mRNAs, preventing premature arrest of transcription. The RNA binding capacity of the N-terminal domain CAT is controlled by phosphorylations of two homologous regulation modules by the phosphotransferase system (PTS). Previous studies on truncated and mutant proteins provided partial insight into the mechanism of signal transduction between the effector and regulatory modules. We report here the conformational and functional investigation on the allosteric activation of full-length LicT. Combining fluorescence anisotropy and NMR, we find a tight correlation between LicT RNA binding capacity and CAT closure upon PTS-mediated phosphorylation and phosphomimetic mutations. Our study highlights fine structural differences between activation processes. Furthermore, the NMR study of full-length proteins points to the back and forth propagation of structural restraints from the RNA binding to the distal regulatory module.

NMR chemical shift backbone assignment of the viral protein P1 encoded by the African Rice Yellow Mottle Virus.

RNA silencing describes a pan-eukaryotic pathway of gene regulation where doubled stranded RNA are processed by the RNAse III enzyme Dicer or homologs. In particular, plants use it as a way to defend themselves against pathogen invasions. In turn, to evade the plant immune response, viruses have developed anti-RNA silencing mechanisms. They may indeed code for proteins called "viral suppressor of RNA silencing" which block the degrading of viral genomic or messenger RNA by the plant. The Rice Mottle Virus is an African virus of the sobemovirus family, which attacks the most productive rice varieties cultivated on this continent. It encodes P1, a cysteine-rich protein described as a potential RNA silencing suppressor. P1 is a 157 amino-acid long protein, characterized by a high propensity to aggregate concomitant with a limited stability with time in the conditions used in structural studies. To overcome this problem, shorter fragments were also studied. This strategy enabled the assignment of more than 90% backbone resonances of P1. This assignment should set the base of future NMR investigation of the protein structure and of its interactions with rice cellular partners.

Pathological Interactions Between Mutant Thyroid Hormone Receptors and Corepressors and Their Modulation by a Thyroid Hormone Analogue with Therapeutic Potential.

BACKGROUND: Thyroid hormone receptors (TRs) are tightly regulated by the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptors. Three conserved corepressor/NR signature box motifs (CoRNR1-3) forming the nuclear receptor interaction domain have been identified in these corepressors. Whereas TRs regulate multiple normal physiological and developmental pathways, mutations in TRs can result in endocrine diseases and be associated with cancers due to impairment of corepressor release. Three mutants that are located in helix H11 of TRs are of special interest: TRα-M388I, a mutant associated with the development of renal clear cell carcinomas (RCCCs), and TRß-Δ430 and TRß-Δ432, two deletion mutants causing resistance to thyroid hormone syndrome. METHODS: Several cell-based and biophysical methods were used to measure the affinity between wild-type and mutant TRα and TRß and all the CoRNR motifs from corepressors to quantify the effects of different thyroid hormone analogues on these interactions. This study was coupled with the measurement of interactions between wild-type and mutant TRs in the context of a heterodimer with RXR to a NCoR fragment in the presence of the same ligands. Structural insights into the binding mode of corepressors to TRs were assessed in parallel by nuclear magnetic resonance spectroscopy. RESULTS: The study shows that TRs interact more avidly with the silencing mediator of retinoic acid and thyroid hormone receptors than with NCoR peptides, and that TRα binds most avidly to S-CoRNR3, whereas TRß binds preferentially to S-CoRNR2. In the studied TR mutants, a transfer of the CoRNR-specificity toward CoRNR1 was observed, coupled with a significant increase in the binding strength. In contrast to 3,5,3'-triiodothyronine (T3), the agonist TRIAC and the antagonist NH-3 were very efficient at dissociating the abnormally strong interactions between mutant TRßs and corepressors. A strong impairment of T3-binding for TRß mutants was shown compared to TRIAC and NH-3 and could explain the different efficiencies of the different ligands in releasing corepressors from the studied TRß mutants. Consequently, TRIAC was found to be more effective than T3 in facilitating coactivator recruitment and decreasing the dominant activity of TRß-Δ430. CONCLUSION: This study helps to clarify the specific interaction surfaces involved in the pathologic phenotype of TR mutants and demonstrates that TRIAC is a potential therapeutic agent for patients suffering from resistance to thyroid hormone syndromes.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo.

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics.

1H, 13C and 15N backbone chemical shift assignments of camelid single-domain antibodies against active state µ-opioid receptor.

Nanobodies are single chain antibodies that have become a highly valuable and versatile tool for biomolecular and therapeutic research. One application field is the stabilization of active states of flexible proteins, among which G-protein coupled receptors represent a very important class of membrane proteins. Here we present the backbone and side-chain assignment of the 1H, 13C and 15N resonances of Nb33 and Nb39, two nanobodies that recognize and stabilize the µ-opioid receptor to opioids in its active agonist-bound conformation. In addition, we present a comparison of their secondary structures as derived from NMR chemical shifts.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

Propagation of conformational changes during µ-opioid receptor activation.

µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the µOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of µOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the µOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the ß2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.

Equivalence between Euler angle conventions for the description of tensorial interactions in liquid NMR: application to different software programs.

Long-range orientational restraints derived from alignment or rotational diffusion tensors have greatly contributed to the expansion of applications in biomolecular NMR. The orientation of the principal axis system of these tensors is usually described by the so-called Euler angles. However, no clear consensus has emerged concerning the convention of the associated orthogonal rotations. As a result, the different programs that derive or predict them have adopted different conventions, which make comparison between their results difficult. Moreover, the rotation schemes are seldom completely described. Here, we summarize the different conventions, determine which ones are adopted by commonly used software packages, and establish the formal equivalencies between the different calculated Euler angles.

Low concentration of a Gd-chelate increases the signal-to-noise ratio in fast pulsing BEST experiments.

Despite numerous developments in the past few years that aim to increase the sensitivity of NMR multidimensional experiments, NMR spectroscopy still suffers from intrinsic low sensitivity. In this report, we show that the combination of two developments in the field, the Band-selective Excitation Short-Transient (BEST) experiment [Schanda et al., J. Am. Chem. Soc., 128 (2006) 9042] and the addition of the nonionic paramagnetic gadolinium chelate gadodiamide into NMR samples, enhances the signal-to-noise ratio. This effect is shown here for four different proteins, three globular and one unfolded, of molecular weights ranging from 6.5 kDa to 40 kDa, using 2D BEST HSQC and 3D BEST triple resonance sequences. Moreover, we show that the increase in signal-to-noise ratio provided by the gadodiamide is higher for peak resonances with lower than average intensity in BEST experiments. It is interesting to note that these residues are on average the weakest ones in those experiments. In this case, the gadodiamide-mediated increase can reach a value of 60% for low and 30% for high molecular weight proteins respectively. An investigation into the origin of this "paramagnetic gain" in BEST experiments is presented.

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.

The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R224₋274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224₋274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹5N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.

Structure of the third intracellular loop of the vasopressin V2 receptor and conformational changes upon binding to gC1qR.

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GalphaS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted alpha-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GalphaS. Moreover, we could estimate the apparent K(d) of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.

Structural mechanism of signal transduction between the RNA-binding domain and the phosphotransferase system regulation domain of the LicT antiterminator.

LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker in-between. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNA-binding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsin-resistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.

The multifunctional protein GC1q-R interacts specifically with the i3 loop arginine cluster of the vasopressin V2 receptor.

In this study, we identified the multifunctional protein GC1q-R as a novel vasopressin V(2) receptor (V(2)R) interacting protein. For this purpose, we have developed a proteomic approach combining pull-down assays using a cyclic peptide mimicking the third intracellular loop of V(2)R as a bait and mass spectrometry analyses of proteins isolated from either rat or human kidney tissues or the HEK 293 cell line. Co-immunoprecipitation of GC1q-R with the c-Myc-tagged h-V(2)R expressed in a HEK cell line confirmed the existence of a specific interaction between GC1q-R and the V(2) receptor. Then, construction of a mutant receptor in i3 loop allowed us to identify the i3 loop arginine cluster of the vasopressin V(2) receptor as the interacting determinant for GC1q-R interaction. Using purified receptor as a bait and recombinant (74-282) GC1q-R, we demonstrated a direct and specific interaction between these two proteins via the arginine cluster.

Rapid determination of protein solubility and stability conditions for NMR studies using incomplete factorial design.

Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters.

Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs.

A ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates.