scholarly journals Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein

2019 ◽  
Vol 476 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Egon Deyaert ◽  
Margaux Leemans ◽  
Ranjan Kumar Singh ◽  
Rodrigo Gallardo ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Conformational Dynamics ◽  
Bound State ◽  
Chlorobium Tepidum ◽  
Leucine Rich Repeat ◽  
Hydrogen Deuterium ◽  
Gtpase Cycle ◽  
Hdx Ms ◽  
First Time

Abstract The LRR (leucine-rich repeat)–Roc (Ras of complex proteins)–COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer–monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5′-diphosphate)-bound states and monomeric in the GTP (guanosine-5′-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR–Roc–COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the ‘Roc dimerization loop’. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

scholarly journals A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD

2021 ◽  
Author(s):  
Loredana Bury ◽  
Emanuela Falcinelli ◽  
Haripriya Kuchi Bhotla ◽  
Anna Maria Mezzasoma ◽  
Giuseppe Guglielmini ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Von Willebrand Disease ◽  
High Affinity ◽  
Laboratory Test Results ◽  
Hydrogen Deuterium ◽  
Von Willebrand ◽  
The Impact ◽  
First Time ◽  
Willebrand Factor

Gain-of-function (GoF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα-von Willebrand factor (VWF) interaction and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GoF GP1BA variants generate a high affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and on GPIbα structure. CHO cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type (WT) GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometryshowed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GoF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα inducing a conformation with high affinity for VWF.

scholarly journals Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruyu Jia ◽  
Chloe Martens ◽  
Mrinal Shekhar ◽  
Shashank Pant ◽  
Grant A. Pellowe ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Allosteric Coupling ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

scholarly journals Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks

2020 ◽  
Author(s):  
Jean Lesne ◽  
Marie Locard-Paulet ◽  
Julien Parra ◽  
Dušan Zivković ◽  
Marie-Pierre Bousquet ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Complexes ◽  
3D Visualization ◽  
Solvent Accessibility ◽  
Proof Of Concept ◽  
Bioinformatic Pipeline ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
First Time ◽  
Structural Insights

AbstractHere, we used for the first time Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28αβ or PA28γ activators. Their solvent accessibility was analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirmed the existence of allosteric differences between the std20S and i20S at the surface of the α-ring triggered from inside the catalytic β-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modified solvent accessibility due to conformational changes of the β-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

scholarly journals Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

2020 ◽  
Author(s):  
Ruyu Jia ◽  
Chloe Martens ◽  
Mrinal Shekhar ◽  
Shashank Pant ◽  
Grant A. Pellowe ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Allosteric Coupling ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

scholarly journals Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marcel J Tauchert ◽  
Jean-Baptiste Fourmann ◽  
Reinhard Lührmann ◽  
Ralf Ficner
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Bound State ◽  
Rna Helicases ◽  
Large Rearrangements ◽  
Rna Complex ◽  
Structural Insights ◽  
Terminal Domains

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.

scholarly journals Activation of GCN2 by the ribosomal P-stalk

2019 ◽  
Vol 116 (11) ◽  
pp. 4946-4954 ◽  
Author(s):  
Alison J. Inglis ◽  
Glenn R. Masson ◽  
Sichen Shao ◽  
Olga Perisic ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

scholarly journals MinE conformational dynamics regulate membrane binding, MinD interaction, and Min oscillation

2017 ◽  
Vol 114 (29) ◽  
pp. 7497-7504 ◽  
Author(s):  
Kyung-Tae Park ◽  
Maria T. Villar ◽  
Antonio Artigues ◽  
Joe Lutkenhaus
Keyword(s):  
Conformational Dynamics ◽  
Active Form ◽  
Hydrogen Deuterium Exchange ◽  
Conformational Switch ◽  
Form Analysis ◽  
Hydrogen Deuterium ◽  
Z Ring ◽  
Hdx Ms ◽  
Β Sheet ◽  
Rule Out

InEscherichia coliMinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6β-stranded form that diffuses in the cytoplasm to an active 4β-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the β-sheet is fine-tuned to balance MinE’s need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.

scholarly journals Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser

2019 ◽  
Vol 73 (8-9) ◽  
pp. 509-518
Author(s):  
Jienv Ding ◽  
Monalisa Swain ◽  
Ping Yu ◽  
Jason R. Stagno ◽  
Yun-Xing Wang
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Free Electron Laser ◽  
Messenger Rna ◽  
Bound States ◽  
Conformational Dynamics ◽  
Thermal Fluctuation ◽  
Binding Pocket ◽  
X Ray ◽  
Aptamer Domain

Abstract Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

scholarly journals Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kaushik Sarkar ◽  
Lisa Joedicke ◽  
Marta Westwood ◽  
Rebecca Burnley ◽  
Michael Wright ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Parathyroid Hormone Receptor ◽  
Exchange Mass Spectrometry ◽  
Gpcr Activation ◽  
Hydrogen Deuterium ◽  
Binding Antibody ◽  
Hdx Ms

Abstract Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

scholarly journals Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

2016 ◽  
Vol 113 (9) ◽  
pp. 2412-2417 ◽  
Author(s):  
Siavash Vahidi ◽  
Yumin Bi ◽  
Stanley D. Dunn ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Molecular Motor ◽  
Conformational Dynamics ◽  
Bound State ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Membrane Bound ◽  
Deuterium Exchange Mass Spectrometry

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

Sign in / Sign up

Export Citation Format

Share Document