Biology subjects

Steyaert, J.

Publications and source records attributed to Steyaert, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Extracellular modulation of TREK-2 activity with nanobodies provides insights into the mechanism of K2P channel regulation

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, we generated camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identified selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. It also facilitated design of a biparatropic inhibitory nanobody with markedly improved affinity and efficacy. These new tools therefore provide important insights into TREK channel gating and suggest alternative, highly selective approach to modulation of K2P channel activity via their extracellular domains.

biophysics↗

bioRxiv2023

Mimicking the regulatory state of a major facilitator superfamily sugar transporter

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N-and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

molecular biology↗

bioRxiv2023

PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

ALS is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of fALS cases. Studying the mechanisms by which mutant FUS is toxic to neurons may provide insight into the pathogenesis of both familial and sporadic forms of ALS. Here we identify Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS in vivo, looking from fly to human. PP2A-C and GSK3{beta} inhibition rescued FUS-induced toxicity in Drosophila and disease-relevant phenotypes in human iPSC-derived spinal motor neurons (sMNs). In both Drosophila and human iPSC-sMNs, we observed reduced GSK3{beta} inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3{beta} hyperactivity. We found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation, and in synergy they modulate mitochondrial transport through the motor protein kinesin. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3 and kinesin in FUS-associated ALS.

neuroscience↗

bioRxiv2023

Structural basis of membrane targeting and coatomer assembly by human GBP1

Guanylate-Binding Proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) that mediate immune effector functions against intracellular pathogens. A key step for the antimicrobial activity of GBPs is the formation of homo- and heterooligomeric complexes on the membrane of pathogen-associated compartments or cytosolinvasive bacteria. Similar to other large GTPases of the dynamin family, oligomerisation and membrane association of GBPs depend on their GTPase activity. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation remains unclear. Here, we report the cryo-EM structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and resolve the molecular ultrastructure of GBP1 coatomer assemblies on liposomes and bacterial lipopolysaccharide membranes. We show how nucleotide binding promotes large-scale conformational changes of the middle and GTPase effector domains that expose the isoprenylated carboxyl-terminus for association with lipid membranes. Our structure reveals how the -helical stalks of the middle domain form a parallel arrangement firmly held in a unique cross-over arrangement by intermolecular contacts between adjacent monomers. This conformation is critical for GBP1 dimers to assemble into densely packed coatomers on target membranes. The extended -helix of the effector domain is flexible and permits intercalation into the dense lipopolysaccharide layer on the outer membrane of gram-negative bacterial pathogens. We show that nucleotide-dependent oligomerisation and GTP hydrolysis yield GBP1 membrane scaffolds with contractile abilities that promote the formation of tubular membrane protrusions and membrane fragmentation. Collectively, our data provide a structural and mechanistic framework for interrogating the molecular basis for GBP1 effector functions in intracellular immunity.

biophysics↗

bioRxiv2023

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

Proton-coupled oligopeptide transporters (POTs) are promiscuous transporters of the Major Facilitator Superfamily, that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several bacterial and mammalian POT homologs have been extensively studied on a structural and molecular level. However, the molecular basis of recognition and transport of the wide range of peptide substrates has remained elusive. Here we present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe if all natural 8400 di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides of a specific subset with compact hydrophobic residues are the best DtpB binders.

biophysics↗

bioRxiv2022

Nanobody CDR3 mimetics enhance SOS1-catalyzed nucleotide exchange on RAS

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active GTP-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14, which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of the native nanobody. Altogether, this study provides the first proof-of-concept that peptides able to functionally modulate a protein-protein interaction can be obtained by structural mimicry of a nanobody paratope.

bioengineering↗

bioRxiv2022

ChILL & DisCO to discover competitive, connective and allosteric Nanobodies that modulate the SOS1-RAS protein-protein interactions and tune the nucleotide exchange rate

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce novel and broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse Nanobodies that either stabilize or disrupt PPIs in a single experiment. We applied ChILL and DisCO to identify competitive, connective or fully allosteric Nanobodies that inhibit or facilitate the formation of the SOS1*RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro and RAS signalling in cellulo. One of these connective Nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as an innovative pharmacophore for extending the repertoire of potential leads.

bioengineering↗

bioRxiv2022

Cryo-EM structure of an atypical proton-coupled peptide transporter: Di- and tripeptide permease C

Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and Tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 [A]. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

biophysics↗

bioRxiv2021

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinsons disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

biochemistry↗

bioRxiv2021

Development of a universal nanobody-binding Fab module for fiducial-assisted cryo- EM studies of membrane proteins

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon CDR grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryo-EM structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facillitated reliable initial 3D structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, humanized, and can be conveniently expressed in E. coli in large amounts, it may not only be useful for structural biology, but also for biomedical applications.

molecular biology↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2021

Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3K{gamma}, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3K{gamma} function is the association of the p110{gamma} catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a heterodimeric PI3K{gamma} complex, p110{gamma}-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its G{beta}{gamma} binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary G{beta}{gamma} binding site in p110{gamma}. Multiple oncogenic mutations mapped to these novel interfaces and enhanced G{beta}{gamma} activation. A nanobody that specifically binds to the p101-G{beta}{gamma} interface blocks activation providing a novel tool to study and target p110{gamma}-p101-specific signaling events in vivo.

biochemistry↗

bioRxiv2021

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3K{gamma}, is a heterodimeric complex composed of a catalytic p110{gamma} subunit bound to a p101 or p84 regulatory subunit. PI3K{gamma} is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K{gamma} single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3K{gamma} regulation and identifies sites that may be exploited for therapeutic development. Highlights- HDX-MS rapidly identifies epitopes of camelid single-chain nanobodies raised against Class IB PI3K complexes, p110{gamma}/p101 and p110{gamma}/p84 - A nanobody targeting p101 improves local resolution in EM studies with p110{gamma}/p101 facilitating structural characterization of the complex - Nanobodies that bind at the interfaces with the lipidated activators Ras and G{beta}{gamma} can prevent activation of p110{gamma}/p101 and p110{gamma}/p84

biochemistry↗

bioRxiv2021

Snapshots of actin and tubulin folding inside the TRiC chaperonin

The integrity of a cells proteome depends on correct folding of polypeptides by chaperonins. The TCP-1 ring chaperonin (TRiC) acts as obligate folder for >10% of cytosolic proteins, including cytoskeletal proteins actin and tubulin. While its architecture and how it recognises folding substrates is emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and co-chaperone (PhLP2A) at different folding stages, for structure determination by cryogenic electron microscopy. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions towards the central space to achieve their folding. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Furthermore, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging atomistic model of client protein folding through TRiC.

biochemistry↗

bioRxiv2021

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS. SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

cell biology↗

bioRxiv2020

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (G)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for G biogenesis and membrane localization2, 3. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)4. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation5. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to G, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gi1 (isoform 1 of G family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of G, disrupting all three switch regions that convey G nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of G. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of G that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes G-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

biochemistry↗

bioRxiv2019

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 [A] resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAA {beta}3 homopentameric receptor bound to its small-molecule agonist histamine.

biochemistry↗

bioRxiv2019

A functionally conserved mechanism of modulation via a vestibule site in pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) belong to a class of ion channels involved in fast synaptic signaling in the central and peripheral nervous systems. Molecules acting as allosteric modulators target binding sites that are remote from the neurotransmitter binding site, but functionally affect coupling of ligand binding to channel opening. Here, we investigated an allosteric binding site in the ion channel vestibule, which has converged from a series of studies on prokaryote and eukaryote channel homologs. We discovered single domain antibodies, called nanobodies, which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote channel ELIC bound either to a positive (PAM) or a negative (NAM) allosteric modulator. We extrapolate the functional importance of the vestibule binding site to eukaryote ion channels, suggesting a conserved mechanism of allosteric modulation. This work identifies key elements of allosteric binding sites and extends drug design possibilities in pLGICs using nanobodies.

neuroscience↗