Mutational analysis to explore long-range allosteric couplings involved in a pentameric channel receptor pre-activation and activation

Pentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood as intermediate states remain poorly characterized by structural approaches. In a previous study on the prototypic bacterial proton-gated channel GLIC, we generated several fluorescent sensors of the protein conformation that report a fast transition to a pre-active state, which precedes the slower process of activation with pore opening. Here, we explored the phenotype of a series of allosteric mutations, using paralleled steady-state fluorescence and electrophysiological measurements over a broad pH range. Our data, fitted to a 3-states Monod-Wyman-Changeux (MWC) model, show that mutations at the subunit interface in the extracellular domain (ECD) principally alter pre-activation, while mutations in the lower ECD and in the transmembrane domain principally alter activation. We also show that propofol alters both transitions. Data are discussed in the framework of transition pathways generated by normal mode analysis (iModFit) that suggest collective protein motions concerted with pore opening. It further supports that pre-activation involves major quaternary compaction of the ECD, and suggests that activation involves principally a re-organization of a 'central gating region' involving a contraction of the ECD β-sandwich and the tilt of the channel lining M2 helix.

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Mutational analysis to explore long-range allosteric coupling and decoupling in a pentameric channel receptor

10.1101/2020.07.03.173997 ◽

2020 ◽

Author(s):

Solène N. Lefebvre ◽

Antoine Taly ◽

Anaïs Menny ◽

Karima Medjebeur ◽

Pierre-Jean Corringer

Keyword(s):

Long Range ◽

Mutational Analysis ◽

Normal Mode Analysis ◽

Mode Analysis ◽

Loss Of Function ◽

Allosteric Coupling ◽

Electrophysiological Recordings ◽

Pore Opening ◽

Conformational Landscape ◽

Ph Range

AbstractPentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood. On the prototypic bacterial channel GLIC, we explored the conformational landscape of the protein during pH-gating. To this aim, we introduced a series of allosteric mutations, and characterized the protein conformation over a broad pH range. We combined electrophysiological recordings, fluorescence quenching experiments monitoring key quaternary reorganizations, and simulations by normal mode analysis. Moderate loss-of-function mutations and the allosteric modulator propofol displace allosteric equilibria involved in pre-activation and pore opening processes, highlighting long-range allosteric coupling between distant regions of the protein. In contrast, total loss-of-function mutations stabilize the protein in unique intermediate conformations where motions are decoupled. Altogether, our data show that the protein can access a wide conformational landscape, raising the possibility of multiple conformational pathways during gating.

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Normal Mode Analysis in Studying Protein Motions with X-Ray Crystallography

Normal Mode Analysis ◽

10.1201/9781420035070-14 ◽

2005 ◽

pp. 179-194

Keyword(s):

Normal Mode ◽

Normal Mode Analysis ◽

Mode Analysis ◽

X Ray ◽

Protein Motions ◽

X Ray Crystallography

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Structural basis for allosteric transitions of a multidomain pentameric ligand-gated ion channel

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922701117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13437-13446 ◽

Cited By ~ 3

Author(s):

Haidai Hu ◽

Rebecca J. Howard ◽

Ugo Bastolla ◽

Erik Lindahl ◽

Marc Delarue

Keyword(s):

Ion Channels ◽

Normal Mode Analysis ◽

Mode Analysis ◽

Structural Basis ◽

State Transitions ◽

Amino Terminal ◽

Subunit Interface ◽

High Resolution Structure ◽

Electrochemical Signal ◽

Multiple Conformations

Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure–function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from aDesulfofustisdeltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca2+with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a “locally closed” conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+modulation and periplasmic NTDs.

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Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013452 ◽

2020 ◽

Vol 295 (33) ◽

pp. 11495-11512 ◽

Cited By ~ 1

Author(s):

Selwyn S. Jayakar ◽

David C. Chiara ◽

Xiaojuan Zhou ◽

Bo Wu ◽

Karol S. Bruzik ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Transmembrane Domain ◽

Mutational Analysis ◽

Photoaffinity Labeling ◽

Subunit Interface ◽

Steroid Sensitivity ◽

Steroid Binding ◽

Definition Of

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+–α− subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+–α− subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.

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Dynamic Neutron Scattering by Biological Systems

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033358 ◽

2018 ◽

Vol 47 (1) ◽

pp. 335-354 ◽

Cited By ~ 12

Author(s):

Jeremy C. Smith ◽

Pan Tan ◽

Loukas Petridis ◽

Liang Hong

Keyword(s):

Neutron Scattering ◽

Normal Mode Analysis ◽

Biological Systems ◽

Coherent Scattering ◽

Dynamics Simulation ◽

Mode Analysis ◽

New Techniques ◽

Protein Motions ◽

Collective Motions ◽

The Rich

Dynamic neutron scattering directly probes motions in biological systems on femtosecond to microsecond timescales. When combined with molecular dynamics simulation and normal mode analysis, detailed descriptions of the forms and frequencies of motions can be derived. We examine vibrations in proteins, the temperature dependence of protein motions, and concepts describing the rich variety of motions detectable using neutrons in biological systems at physiological temperatures. New techniques for deriving information on collective motions using coherent scattering are also reviewed.

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Predicting protein functional motions: an old recipe with a new twist

10.1101/703652 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sergei Grudinin ◽

Elodie Laine ◽

Alexandre Hoffmann

Keyword(s):

Normal Mode ◽

Conformational Changes ◽

Normal Mode Analysis ◽

Normal Modes ◽

Low Frequency ◽

Structural Heterogeneity ◽

Mode Analysis ◽

Protein Motions ◽

Wide Range ◽

Geometrical Constraints

Large macromolecules, including proteins and their complexes, very often adopt multiple conformations. Some of them can be seen experimentally, for example with X-ray crystallography or cryo-electron microscopy. This structural heterogeneity is not occasional and is frequently linked with specific biological function. Thus, the accurate description of macromolecular conformational transitions is crucial for understanding fundamental mechanisms of life’s machinery. We report on a real-time method to predict such transitions by extrapolating from instantaneous eigen-motions, computed using the normal mode analysis, to a series of twists. We demonstrate the applicability of our approach to the prediction of a wide range of motions, including large collective opening-closing transitions and conformational changes induced by partner binding. We also highlight particularly difficult cases of very small transitions between crystal and solution structures. Our method guaranties preservation of the protein structure during the transition and allows to access conformations that are unreachable with classical normal mode analysis. We provide practical solutions to describe localized motions with a few low-frequency modes and to relax some geometrical constraints along the predicted transitions. This work opens the way to the systematic description of protein motions, whatever their degree of collectivity. Our method is available as a part of the NOn-Linear rigid Block (NOLB) package at https://team.inria.fr/nano-d/software/nolb-normal-modes/.Significance StatementProteins perform their biological functions by changing their shapes and interacting with each other. Getting access to these motions is challenging. In this work, we present a method that generates plausible physics-based protein motions and conformations. We model a protein as a network of atoms connected by springs and deform it along the least-energy directions. Our main contribution is to perform the deformations in a nonlinear way, through a series of twists. This allows us to produce a wide range of motions, some of them previously inaccessible, and to preserve the structure of the protein during the motion. We are able to simulate the opening or closing of a protein and the changes it undergoes to adapt to a partner.

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Normal Mode Analysis in Studying Protein Motions with X-Ray Crystallography

Normal Mode Analysis - Chapman & Hall/CRC Mathematical & Computational Biology ◽

10.1201/9781420035070.ch8 ◽

2005 ◽

pp. 155-169

Author(s):

George Phillips

Keyword(s):

Normal Mode ◽

Normal Mode Analysis ◽

Mode Analysis ◽

X Ray ◽

Protein Motions ◽

X Ray Crystallography

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Application of the ESMACS Binding Free Energy Protocol to a Highly Varied Ligand Dataset: Lactate Dehydogenase A

10.26434/chemrxiv.8398055 ◽

2019 ◽

Author(s):

David Wright ◽

Fouad Husseini ◽

Shunzhou Wan ◽

Christophe Meyer ◽

Herman Van Vlijmen ◽

...

Keyword(s):

Free Energy ◽

Surface Area ◽

Binding Free Energy ◽

Normal Mode Analysis ◽

Binding Mode ◽

Accessible Surface Area ◽

Solvent Accessible Surface Area ◽

Mode Analysis ◽

Energy Calculation ◽

Accessible Surface

<div>Here, we evaluate the performance of our range of ensemble simulation based binding free energy calculation protocols, called ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) for use in fragment based drug design scenarios. ESMACS is designed to generate reproducible binding affinity predictions from the widely used molecular mechanics Poisson-Boltzmann surface area (MMPBSA) approach. We study ligands designed to target two binding pockets in the lactate dehydogenase A target protein, which vary in size, charge and binding mode. When comparing to experimental results, we obtain excellent statistical rankings across this highly diverse set of ligands. In addition, we investigate three approaches to account for entropic contributions not captured by standard MMPBSA calculations: (1) normal mode analysis, (2) weighted solvent accessible surface area (WSAS) and (3) variational entropy. </div>

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RAMAN SPECTRA OF TITANIUM DI-, TRI-, AND TETRA-CHLORIDES

International Journal of Modern Physics B ◽

10.1142/s021797920100886x ◽

2001 ◽

Vol 15 (28n30) ◽

pp. 3865-3868 ◽

Cited By ~ 2

Author(s):

H. MIYAOKA ◽

T. KUZE ◽

H. SANO ◽

H. MORI ◽

G. MIZUTANI ◽

...

Keyword(s):

Force Constant ◽

Raman Spectra ◽

Normal Mode ◽

Raman Band ◽

Normal Mode Analysis ◽

Force Constants ◽

Mode Analysis ◽

Oxidation Number

We have obtained the Raman spectra of TiCl n (n= 2, 3, and 4). Assignments of the observed Raman bands were made by a normal mode analysis. The force constants were determined from the observed Raman band frequencies. We have found that the Ti-Cl stretching force constant increases as the oxidation number of the Ti species increases.

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