Biology subjects

Laurents, D. V.

Publications and source records attributed to Laurents, D. V..

14 recordsLinked to original sources

bioRxiv2025

Pathological mutation in SMN impairs modulation of GAR1 phase separation linking condensate dysfunction to Spinal Muscular Atrophy

Pathological mutations in liquid-liquid phase separation (LLPS) scaffold proteins have been linked to biomolecular condensate dysfunction in neurodegenerative diseases, while the possible impact of client protein mutations remains unclear. In spinal muscular atrophy (SMA), the disease-associated E134K mutation in the Tudor domain (TD) of the survival motor neuron (SMN) protein disrupts its interaction with GAR1, an RGG-rich protein within the H/ACA small nucleolar ribonucleoprotein complex. The consequences of this impaired interaction have not been elucidated. Here, we identified GAR1 as an LLPS scaffold protein that is phase separated in nuclear compartments and forms gel-like condensates via complex coacervation with RNA in vitro. In cells, we reveal that SMN co-localizes with GAR1 in Cajal bodies and modulates its dynamics. Using confocal microscopy and NMR spectroscopy, we further show that SMN TD is a client protein that regulates the architecture and dynamics of GAR1 condensates through competitive RNA interactions, implicating GAR1 phase separation in RNA accumulation and release processes regulated by SMN. Notably, the SMA-associated E134K variant of SMN exhibits a reduced affinity for GAR1, impairing the modulation of GAR1 condensates and displacement RNA. Our findings suggest a mechanistic link between phase separation dysregulation and SMA, driven by disrupted scaffold-client interactions, that highlights the therapeutic potential of targeting SMN-dependent condensate regulation in SMA.

biochemistry↗

bioRxiv2024

The C-terminal tails of GroEL and its mitochondrial and chloroplastic homologs adopt polyproline II helices.

The chaperonin GroEL and its mitochondrial and chloroplastic homologs mHsp60 and Cpn60 are large barrel-like oligomeric proteins. Chaperonins facilitate folding by isolating nascent chains in their hollow interior and undergoing conformational transitions driven by ATP hydrolysis. Due to their vital importance, the structure of GroEL and its homologs have been extensively studied by X-ray crystallography and CryoEM, revealing one or two rings each of which contains seven subunits. Each subunit has three folded domains and a twenty-four residue C-terminal extension. Whereas this C-terminal tail has been reported to bind and stimulate the folding of the client protein, it appears to be invisible or blurry, which suggests disorder. The objective of this study is to characterize conformational preferences in the C-terminal tails of GroEL, mHsp60 and representative Cpn60s using circular dichroism and nuclear magnetic resonance spectroscopies. The tails of GroEL and mHsp60 consist of two segments. The first is rich in residues typical of intrinsically disordered proteins and the second segment consists exclusively (GroEL) or almost entirely (mHsp60) of Gly and Met residues. The spectroscopic results evince that these C-terminal extensions are not wholly disordered but adopt polyproline II helices whose populations are higher in the second Gly/Met-rich segment. Whereas the C-terminal segments of chloroplastic chaperonins are Gly-poor, they are rich in proline and also adopt polyproline II helix conformations. These results provide insight into the function of chaperonin C-terminal tails.

biochemistry↗

bioRxiv2023

Architectonic Principles of Polyproline II Bundle Protein Domains

Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of -helices and {beta}-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of C-H{middle dot}{middle dot}{middle dot}O=C hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins.

biochemistry↗

bioRxiv2022

Insight into Polyproline II Helical Bundle Stability and Folding from NMR Spectroscopic Characterization of the Snow Flea Antifreeze Protein Denatured State

The use of PPII helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. To get atomic level information on the conformational ensemble and entropy of the reduced denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at three experimental conditions. No significant populations of preferred secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation of the two disulfide bonds during nascent folding. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured /{beta} proteins both on fast ps/ns as well as slower s/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

biophysics↗

bioRxiv2022

Fused in Sarcoma undergoes cold denaturation: implications on phase separation

The mediation of fused in sarcoma (FUS) protein liquid-liquid phase separation (LLPS) is generally attributed to the low-complexity and disordered domains, while the role of its folded domains remains unknown. In this work we questioned the role of the folded domains on the full-length (FL) FUS LLPS and studied the influence of several metabolites, ions and overall conditions on the LLPS process using turbidity assays, differential interference contrast microscopy and nuclear magnetic resonance spectroscopy. We demonstrate that FL FUS LLPS is highly responsive to the surrounding conditions, and that overall intrinsic disorder is crucial for LLPS. To promote such disorder, we reveal that the FUS RNA-recognition domain (RRM) and the zinc-finger motif (ZnF) undergo cold denaturation above 0{o}C, at a temperature that is determined by the conformational stability of the ZnF domain. We hypothesize that, in cold shock conditions, cold denaturation might provide a pathway that exposes additional residues to promote FUS self-assembly. Such findings mark the first evidence that FUS globular domains may have an active role in stress granule formation in cold stress.

biophysics↗

bioRxiv2021

SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements

The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long -helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the -helix population progressively declines towards the C-termini, and dynamics measurements indicate that the Nsp8 NTD behaves as a dimer under our conditions.

biophysics↗

bioRxiv2020

Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to "hack" these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13C,15N-labeled C-terminal region of Nsp2. The 13C{beta} and backbone 13CO, 1HN, 13C and 15N nuclei were assigned by analysis of a series of 2D 1H-15N HSQC and 13C-15N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of {beta}-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T1{rho} measurements, the {1H}-15N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.

biophysics↗

bioRxiv2020

Glycine Rich Segments Adopt Polyproline II Helices Which May Contribute to Biomolecular Condensate Formation

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Considering NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. In cells, the polyproline II population of these peptides could be augmented by binding profilin protein or SH3, WW or OCRE domains, association with RNA or assembly into polyproline II helical bundles. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

biophysics↗

bioRxiv2020

Tau amyloidogenesis begins with a loss of its conformational polymorphism

Knowledge on the molecular bases of early amyloid assembly is fundamental to understand its structure-dysfunction relationship during disease progression. Tauopathies, a well-defined set of neurodegenerative disorders that includes Alzheimers disease, are characterized by the pathological amyloid aggregation of tau. However, the underlying molecular mechanisms that trigger tau aggregation and toxicity are poorly understood. Here, using a single-molecule approach, AFM-based single molecule-force spectroscopy (AFM-SMFS), combined with a protein-engineering mechanical protection strategy, we have analyzed the fluctuations of the conformational space of tau during the start of its pathological amyloid assembly. Specifically, we have analyzed the region that includes the four tau microtubule-binding repeats, known to play a key role on tau aggregation. We find that, unlike other amyloid-forming proteins, tau aggregation is accompanied by a decrease of conformational polymorphism, which is driven by amyloid-promoting factors, such as the {Delta}280K and P301L mutations, linked to Frontotemporal Dementia-17, or by specific chemical conditions. Such perturbations have distinct effects and lead to different tau (aggregate) structures. In addition to providing insight into how tau aggregates in a context dependent manner, these findings may help delve into how protein aggregation-based diseases, like Alzheimers, might be treated using monomer fluctuations as a pharmacological target. O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/158923v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@951f1borg.highwire.dtl.DTLVardef@2d5f5borg.highwire.dtl.DTLVardef@cf7776org.highwire.dtl.DTLVardef@abe671_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Molecular determinants of liquid demixing and amyloidogenesis in human CPEB3

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), is an RNA-binding protein which in its soluble state is localized in membraneless neuronal RNA granules keeping target mRNAs in a repressed state. The stimulus-dependent aggregation of CPEB3 activates target mRNAs translation, a central event for the maintenance of long-term memory-related synaptic plasticity in mammals. To date, the molecular determinants that govern both connected events remain unclear. Here, to gain insight into these processes, the biophysical properties of the human CPEB3 (hCPEB3) are characterized. We found that hCPEB3 homotypic condensation is mainly driven by hydrophobic interactions and occurs under physiological conditions. Moreover, hCPEB3 biomolecular condensates are dynamic inside living cells, whose localization and stabilization are mediated by its RNA-recognition domains. In contrast, the hCPEB3 polar N-terminal region is crucial for hCPEB3 amyloid-like aggregation in vitro, which is disrupted by the polyglutamine binding peptide 1 (QBP1), A{beta}42 seeds and Hsp70, highlighting the importance of the Q4RQ4 tract as well as the hydrophobic residues for hCPEB3 functional aggregation. Based on these findings, we postulate a model for hCPEB3s role in memory persistence that advances a rather sophisticated control for hCPEB3 condensate dissociation and amyloid-like formation to achieve its physiological function. HighlightsO_LIhCPEB3 forms toxic intermediates that persist longer than in other functional amyloids. C_LIO_LIRNA-recognition domains stabilize hCPEB3 granule formation and dynamics. C_LIO_LIDifferent segments within hCPEB3 promote amyloidogenesis and liquid demixing. C_LIO_LIhCPEB3 amyloid formation requires both hydrophobic and polyQ segments. C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

neuroscience↗

bioRxiv2020

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

Functional amyloids are present in a wide variety of organisms ranging from bacteria to humans. Experience-dependent aggregation of the cytoplasmic polyadenylation element-binding (CPEB) prion-like protein to a translationally active state has emerged as a plausible biochemical substrate of long-lasting memories. CPEB aggregation is driven by prion-like domains (PLD) that are highly divergent in sequence across species. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD in vitro using single-molecule and bulk biophysical methods and compare them with those previously reported for neuronal Drosophila CPEB, Orb2 PLD. The existence of transient oligomers and mature filaments suggests similarities in the late stages of the assembly pathway for both PLDs. However, while prior to aggregation the Orb2 PLD monomer remains as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising -helical structures that evolve to coiled-coil species, suggesting structural differences at the beginning of their amyloid assembly pathways. Our results show how divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

neuroscience↗

bioRxiv2020

Preferred Conformations in the Intrinsically Disordered Region of Human CPEB3 Explain its Role in Memory Consolidation

While implicated in neurodegenerative diseases, amyloids are also essential to some physiological processes, including memory consolidation by neuronal-specific isoforms of the Cytoplasmic Polyadenylation Element Binding (CPEB) protein family. CPEB mediates memory persistence by the formation of self-sustaining amyloid assemblies through its intrinsically disordered region (IDR). Here, we characterize the atomic level conformation and ps-ns dynamics of the 426-residue IDR of human CPEB3 (hCPEB3), which has been associated with episodic memory in humans, by NMR spectroscopy. We found that the first 29 residues: M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical+disordered motif. Residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. While the (VG)5 repeat motif is completely disordered, residues 200-250 adopt three partially populated -helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated -helix and border a phosphoTyr which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Besides revealing some aspects of the molecular basis of memory, these findings could aid the future development of treatments for post-traumatic stress disorder. Areas: Biophysics, Structural Biology, Biochemistry & Neurosciences.

biochemistry↗

bioRxiv2020

Phe-Gly Motifs Drive Fibrillization of TDP-43's Prion-Like Domain Droplets

TDP-43 assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g. muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct endpoint species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs, and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions, and illustrate structural features that distinguishes between aggregate forms.

biophysics↗

bioRxiv2019

smFRET Detects Dual Binding Modes Modulated by Proline Isomerization in a Mega-Dalton Multi-Enzyme Complex

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗