A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

Structure-function relationships in protein homorepeats.

Homorepeats (or polyX), protein segments containing repetitions of the same amino acid, are abundant in proteomes from all kingdoms of life and are involved in crucial biological functions as well as several neurodegenerative and developmental diseases. Mainly inserted in disordered segments of proteins, the structure/function relationships of homorepeats remain largely unexplored. In this review, we summarize present knowledge for the most abundant homorepeats, highlighting the role of the inherent structure and the conformational influence exerted by their flanking regions. Recent experimental and computational methods enable residue-specific investigations of these regions and promise novel structural and dynamic information for this elusive group of proteins. This information should increase our knowledge about the structural bases of phenomena such as liquid-liquid phase separation and trinucleotide repeat disorders.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction.

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the ß2-adernergic receptor (ß2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies.

Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems.

Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1.

Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

eIF4G1 N-terminal intrinsically disordered domain is a multi-docking station for RNA, Pab1, Pub1, and self-assembly.

Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the N-terminal intrinsically disordered region of eIF4G1 (residues 1-249) and its interactions with Pub1, Pab1 and RNA. The conformational ensemble of eIF4G11-249 shows an α-helix within the BOX3 conserved element and a dynamic network of fuzzy π-π and π-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. The RNA1 region interacts with RNA through a new RNA interaction motif and with the Pub1 RRM3 domain. This later also interacts with eIF4G1 BOX1 modulating its intrinsic self-assembly properties. The description of the biomolecular interactions involving eIF4G1 to the residue detail increases our knowledge about biological processes involving this key translation initiation factor.

Structure of the vasopressin hormone-V2 receptor-ß-arrestin1 ternary complex.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with ß-arrestin1. It reveals an atypical position of ß-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/ß-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R carboxyl terminus are clearly identified and interact extensively with the ß-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a notable structural variability among GPCR-arrestin signaling complexes.

Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction.

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the ß2-adernergic receptor (ß2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

Δ133p53ß isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells.

The p53 isoform, Δ133p53ß, is critical in promoting cancer. Here we report that Δ133p53ß activity is regulated through an aggregation-dependent mechanism. Δ133p53ß aggregates were observed in cancer cells and tumour biopsies. The Δ133p53ß aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53ß aggregates and loss of Δ133p53ß dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53ß from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion.

The diversity of molecular interactions involving intrinsically disordered proteins: A molecular modeling perspective.

Intrinsically Disordered Proteins and Regions (IDPs/IDRs) are key components of a multitude of biological processes. Conformational malleability enables IDPs/IDRs to perform very specialized functions that cannot be accomplished by globular proteins. The functional role for most of these proteins is related to the recognition of other biomolecules to regulate biological processes or as a part of signaling pathways. Depending on the extent of disorder, the number of interacting sites and the type of partner, very different architectures for the resulting assemblies are possible. More recently, molecular condensates with liquid-like properties composed of multiple copies of IDPs and nucleic acids have been proven to regulate key processes in eukaryotic cells. The structural and kinetic details of disordered biomolecular complexes are difficult to unveil experimentally due to their inherent conformational heterogeneity. Computational approaches, alone or in combination with experimental data, have emerged as unavoidable tools to understand the functional mechanisms of this elusive type of assemblies. The level of description used, all-atom or coarse-grained, strongly depends on the size of the molecular systems and on the timescale of the investigated mechanism. In this mini-review, we describe the most relevant architectures found for molecular interactions involving IDPs/IDRs and the computational strategies applied for their investigation.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR).

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins.

The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.

Predicting Secondary Structure Propensities in IDPs Using Simple Statistics from Three-Residue Fragments.

Intrinsically disordered proteins (IDPs) play key functional roles facilitated by their inherent plasticity. In most of the cases, IDPs recognize their partners through partially structured elements inserted in fully disordered chains. The identification and characterization of these elements is fundamental to understand the functional mechanisms of IDPs. Although several computational methods have been developed to identify order within disordered chains, most of the current secondary structure predictors are focused on globular proteins and are not necessarily appropriate for IDPs. Here, we present a comprehensible method, called Local Structural Propensity Predictor (LS2P), to predict secondary structure elements from IDP sequences. LS2P performs statistical analyses from a database of three-residue fragments extracted from coil regions of high-resolution protein structures. In addition to identifying scarcely populated helical and extended regions, the method pinpoints short stretches triggering ß-turn formation or promoting α-helices. The simplicity of the method enables a direct connection between experimental observations and structural features encoded in IDP sequences.

IDPs and their complexes in GPCR and nuclear receptor signaling.

G protein-coupled receptors (GPCRs) and Nuclear Receptors (NRs) are two signaling machineries that are involved in major physiological processes and, as a consequence, in a substantial number of diseases. Therefore, they actually represent two major targets for drugs with potential applications in almost all public health issues. Full exploitation of these targets for therapeutic purposes nevertheless requires opening original avenues in drug design, and this in turn implies a better understanding of the molecular mechanisms underlying their functioning. However, full comprehension of how these complex systems function and how they are deregulated in a physiopathological context is obscured by the fact that these proteins include a substantial number of disordered regions that are central to their mechanism of action but whose structural and functional properties are still largely unexplored. In this chapter, we describe how these intrinsically disordered regions (IDR) or proteins (IDP) intervene, control and finely modulate the thermodynamics of complexes involved in GPCR and NR regulation, which in turn triggers a multitude of cascade of events that are exquisitely orchestrated to ultimately control the biological output.

Flanking Regions Determine the Structure of the Poly-Glutamine in Huntingtin through Mechanisms Common among Glutamine-Rich Human Proteins.

The causative agent of Huntington's disease, the poly-Q homo-repeat in the N-terminal region of huntingtin (httex1), is flanked by a 17-residue-long fragment (N17) and a proline-rich region (PRR), which promote and inhibit the aggregation propensity of the protein, respectively, by poorly understood mechanisms. Based on experimental data obtained from site-specifically labeled NMR samples, we derived an ensemble model of httex1 that identified both flanking regions as opposing poly-Q secondary structure promoters. While N17 triggers helicity through a promiscuous hydrogen bond network involving the side chains of the first glutamines in the poly-Q tract, the PRR promotes extended conformations in neighboring glutamines. Furthermore, a bioinformatics analysis of the human proteome showed that these structural traits are present in many human glutamine-rich proteins and that they are more prevalent in proteins with longer poly-Q tracts. Taken together, these observations provide the structural bases to understand previous biophysical and functional data on httex1.

Evidence of the Reduced Abundance of Proline cis Conformation in Protein Poly Proline Tracts.

Proline is found in a cis conformation in proteins more often than other proteinogenic amino acids, where it influences structure and modulates function, being the focus of several high-resolution structural studies. However, until now, technical and methodological limitations have hampered the site-specific investigation of the conformational preferences of prolines present in poly proline (poly-P) homorepeats in their protein context. Here, we apply site-specific isotopic labeling to obtain high-resolution NMR data on the cis/trans equilibrium of prolines within the poly-P repeats of huntingtin exon 1, the causative agent of Huntington's disease. Screening prolines in different positions in long (poly-P11) and short (poly-P3) poly-P tracts, we found that, while the first proline of poly-P tracts adopts similar levels of cis conformation as isolated prolines, a length-dependent reduced abundance of cis conformers is observed for terminal prolines. Interestingly, the cis isomer could not be detected in inner prolines, in line with percentages derived from a large database of proline-centered tripeptides extracted from crystallographic structures. These results suggest a strong cooperative effect within poly-Ps that enhances their stiffness by diminishing the stability of the cis conformation. This rigidity is key to rationalizing the protection toward aggregation that the poly-P tract confers to huntingtin. Furthermore, the study provides new avenues to probe the structural properties of poly-P tracts in protein design as scaffolds or nanoscale rulers.

Site-Specific Isotopic Labeling (SSIL): Access to High-Resolution Structural and Dynamic Information in Low-Complexity Proteins.

Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo-electron microscopy, and modern high-field NMR magnets have challenged the limits of high-resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low-complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site-specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell-free protein synthesis to overcome these limitations. An overview on how poly-glutamine tracts were made amenable to high-resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid-liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL.

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression.

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.