scholarly journals Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating

2017 ◽  
Vol 114 (14) ◽  
pp. 3768-3773 ◽  
Author(s):  
Sabrina Vullo ◽  
Gaetano Bonifacio ◽  
Sophie Roy ◽  
Niklaus Johner ◽  
Simon Bernèche ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Ph Sensing ◽  
Central Channel ◽  
Ph Sensors ◽  
Pain Sensation ◽  
Acid Sensing Ion Channels ◽  
Acidic Residues ◽  
Ph Dependent

Acid-sensing ion channels (ASICs) are proton-activated Na+ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

scholarly journals Correlation of membrane protein conformational and functional dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Joel José Montalvo‐Acosta ◽  
George R. Heath ◽  
Yining Jiang ◽  
Xiaolong Gao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
High Temporal Resolution ◽  
Dependent Manner ◽  
Functional Dynamics ◽  
Ph Dependent ◽  
Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

scholarly journals Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sabrina Vullo ◽  
Nicolas Ambrosio ◽  
Jan P Kucera ◽  
Olivier Bignucolo ◽  
Stephan Kellenberger
Keyword(s):  
Ion Channels ◽  
Kinetic Analysis ◽  
Conformational Changes ◽  
Transmembrane Helix ◽  
Activation Mechanism ◽  
Pain Sensation ◽  
Channel Activation ◽  
Channel Pore ◽  
Acid Sensing Ion Channels ◽  
Channel Gate

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

scholarly journals Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor

2015 ◽  
Vol 291 (4) ◽  
pp. 1817-1825 ◽  
Author(s):  
Benjamin T. Walters ◽  
Pernille F. Jensen ◽  
Vincent Larraillet ◽  
Kevin Lin ◽  
Thomas Patapoff ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Hydrogen Exchange ◽  
Dissociation Constants ◽  
Conformational Dynamics ◽  
Fc Receptor ◽  
Salt Bridges ◽  
Neonatal Fc Receptor ◽  
Intermolecular Contacts ◽  
Ph Dependent

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.

NMR and EPR studies of membrane transporters

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Ute A. Hellmich ◽  
Clemens Glaubitz
Keyword(s):  
Membrane Proteins ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Conformational Dynamics ◽  
Crystallographic Analysis ◽  
Membrane Transporters ◽  
Paramagnetic Resonance ◽  
Substrate Protein ◽  
Electron Paramagnetic

AbstractIn order to fulfill their function, membrane transport proteins have to cycle through a number of conformational and/or energetic states. Thus, understanding the role of conformational dynamics seems to be the key for elucidation of the functional mechanism of these proteins. However, membrane proteins in general are often difficult to express heterologously and in sufficient amounts for structural studies. It is especially challenging to trap a stable energy minimum, e.g., for crystallographic analysis. Furthermore, crystallization is often only possible by subjecting the protein to conditions that do not resemble its native environment and crystals can only be snapshots of selected conformational states. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy are complementary methods that offer unique possibilities for studying membrane proteins in their natural membrane environment and for investigating functional conformational changes, lipid interactions, substrate-lipid and substrate-protein interactions, oligomerization states and overall dynamics of membrane transporters. Here, we review recent progress in the field including studies from primary and secondary active transporters.

scholarly journals The complex role of the N-terminus and acidic residues of HdeA as pH-dependent switches in its chaperone function

2020 ◽  
Vol 264 ◽  
pp. 106406 ◽  
Author(s):  
Sayuri Pacheco ◽  
Marlyn A. Widjaja ◽  
Jafaeth S. Gomez ◽  
Karin A. Crowhurst ◽  
Ravinder Abrol
Keyword(s):  
Chaperone Function ◽  
N Terminus ◽  
Acidic Residues ◽  
Ph Dependent

scholarly journals Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae

2018 ◽  
Vol 115 (27) ◽  
pp. E6182-E6190 ◽  
Author(s):  
Derek P. Claxton ◽  
Kevin L. Jagessar ◽  
P. Ryan Steed ◽  
Richard A. Stein ◽  
Hassane S. Mchaourab
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Spin Labels ◽  
Release Mechanism ◽  
Conserved Residues ◽  
Terminal Domain ◽  
Mate Transporter

Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+- and H+-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na+- and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism.

scholarly journals Role of Electrostatic Repulsion in Controlling pH-Dependent Conformational Changes of Viral Fusion Proteins

Structure ◽  
2013 ◽  
Vol 21 (7) ◽  
pp. 1085-1096 ◽  
Author(s):  
Joseph S. Harrison ◽  
Chelsea D. Higgins ◽  
Matthew J. O’Meara ◽  
Jayne F. Koellhoffer ◽  
Brian A. Kuhlman ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fusion Proteins ◽  
Electrostatic Repulsion ◽  
Viral Fusion ◽  
Viral Fusion Proteins ◽  
Ph Dependent

scholarly journals Electrostatic effects in proteins are governed by pH-redistribution of the conformational ensemble

2020 ◽  
Author(s):  
Christos M. Kougentakis ◽  
Ananya Majumdar ◽  
E. Bertrand García-Moreno
Keyword(s):  
Conformational Changes ◽  
De Novo ◽  
Energy Transduction ◽  
Conformational Ensemble ◽  
Biological Macromolecules ◽  
Ph Sensing ◽  
Functional Sites ◽  
Ionizable Residues ◽  
Open States

The imperative for charges to be hydrated is one of the most important organizing principles in biology, responsible for the general architecture of biological macromolecules and for energy storage in the form of electrochemical gradients. Paradoxically, many functional sites in proteins have buried ionizable groups1. These groups are tolerated because they are usually buried in the neutral state2. However, when they become charged they can drive structural transitions to open states in which the charge can be stabilized, mostly through interactions with water3. This coupling between the ionization of a buried group and conformational reorganization is precisely the mechanism used by proteins to perform energy transduction4,5,6. By applying this principle to a family of 25 variants of staphylococcal nuclease with internal Lys residues, it was possible to characterize in detail the range of localized partial unfolding events that even a highly stable protein that unfolds cooperatively can undergo in response to H+-binding. Conformational states that constitute vanishingly small populations of the equilibrium native state ensemble of this protein were identified by correlation of structural and thermodynamic data, providing a map of the conformational landscape of this protein with unprecedented detail. The data demonstrate that the apparent pKa values of buried ionizable residues are not determined by the properties of their microenvironment but by the intrinsic propensity of the protein to populate open states in which internal charged residues can be hydrated. The role of buried residues in functional sites in proteins relies on their ability to tune the conformational ensemble for redistribution in response to small changes in pH. These results provide the physical framework necessary for understanding the role of pH-driven conformational changes in driving biological energy transduction4, the identification of pH-sensing proteins in nature7, and for the engineering of pH-sensitive dynamics and function in de novo designed proteins8.

scholarly journals How do brassinosteroids activate their receptors?

2019 ◽  
Author(s):  
Alexander S. Moffett ◽  
Diwakar Shukla
Keyword(s):  
Conformational Changes ◽  
Functional Role ◽  
Conformational Dynamics ◽  
Growth Hormones ◽  
Dynamics Simulation ◽  
Leucine Rich Repeat ◽  
Plant Growth Hormones ◽  
Simulation Based ◽  
Large Conformational Change

AbstractBrassinosteroids (BRs) are an important class of plant growth hormones which signal through BRI1 and BAK1, leucine-rich repeat receptor-like kinases (LRR-RLKs). When bound to the BRI1 island domain, BRs act as a “molecular glue”, mediating interactions between BRI1 and BAK1 extracellular domains. However, it is unclear how much other factors contribute to BR-induced BRI1-BAK1 association, including stabilization of the BRI1 island domain and large conformational changes in BRI1. We use several molecular dynamics simulation-based methods to explore the contributions of each mechanism to BR-dependent Arabidopsis thaliana BRI1-BAK1 association. We find that specific BR interactions make major contributions to BRI1-BAK1 association free energy. BR binding stabilizes the BRI1 island domain, while BRI1 undergoes a large conformational change to form a secondary interface with BAK1. These results suggest that each mechanism plays a part in BR signal transduction while raising questions about the functional role of conformational dynamics in other LRR-RLKs.

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