Cryo-EM structures of the human sodium-potassium pump revealing the gating mechanism on the cytoplasmic side

2021 ◽  
Author(s):  
Yingying Guo ◽  
Yuanyuan Zhang ◽  
Renhong Yan ◽  
Bangdong Huang ◽  
Fangfei Ye ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Structural Basis ◽  
Extracellular Potassium ◽  
Ion Pump ◽  
Sodium Potassium ◽  
Working Cycle ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Membrane Bound ◽  
Cytoplasmic Side

Abstract Na+/K+-ATPase (NKA) is a membrane-bound ion pump that generates electrochemical gradient of sodium ion and potassium ion across the plasma membrane via hydrolyzing ATP. During each so-called Post-Albers cycle, NKA exchanges three cytoplasmic sodium ions for two extracellular potassium ions through alternating E1 and E2 states. Hitherto, there are several steps remained unknown during the complete working cycle of NKA. Here, we report cryo-electron microscopy (cryo-EM) structures of recombinant over-expressed human NKA in three distinct states at 3.1–3.4 Å resolution, representing the E1·3Na state, in which the cytosolic gate is open, and the E1·3Na·ATP state preceding ATP hydrolysis and a basic E2·[2K] state. These structures reveal the ATP-dependent Na+-binding site remodeling for the close of the cytoplasmic gate, filling a gap in the structural elucidation of the Post-Albers cycle of NKA and providing structural basis for understanding the cytoplasmic Na+ entrance pathway.

Structure-function studies of the sodium pump

1999 ◽  
Vol 77 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rhoda Blostein
Keyword(s):  
Sodium Pump ◽  
Atp Hydrolysis ◽  
Cation Binding ◽  
Structural Basis ◽  
Animal Cells ◽  
Sodium Potassium ◽  
Membrane Protein Complex ◽  
And Function ◽  
P Type ◽  
Hydrolysis Of

The Na+,K+-ATPase is an ubiquitous plasma membrane protein complex that belongs to the P-type family of ion motive ATPases. Under normal conditons, it couples the hydrolysis of one molecule of ATP to the exchange of three Na+ for two K+ ions, thus maintaining the normal gradient of these cations in animal cells. Despite decades of investigation of its structure and function, the structural basis for its cation specificity and for conformational coupling of the scalar energy of ATP hydrolysis to the vectorial movement of Na+ and K+ have remained a major unresolved issue. This paper summarizes our recent studies concerned with these issues. The findings indicate that regions(s) of the amino terminus and first cytoplasmic (M2/M3) loop act synergisticaly to affect the steady-state conformational equilibrium of the enzyme. Although carboxyl- or hydroxyl-bearing amino acids comprise the cation-binding and occlusion sites, our experiments also suggest that these interactions may be modulated by juxtapositioned cytoplasmic regions.Key words: sodium, potassium, ATPase, Na+,K+-ATPase, sodium pump.

scholarly journals Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

2021 ◽  
Author(s):  
Koichiro E. Kishi ◽  
Yoon Seok Kim ◽  
Masahiro Fukuda ◽  
Tsukasa Kusakizako ◽  
Elina Thadhani ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Action Spectrum ◽  
Light Sensitivity ◽  
Structural Basis ◽  
Ion Pump ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Computational Analyses ◽  
Insight Into

ChRmine, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 Å resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

scholarly journals Gating mechanism of cardiac ryanodine receptor 2 upon calcium ion binding

2020 ◽  
Author(s):  
Takuya Kobayashi ◽  
Akihisa Tsutsumi ◽  
Nagomi Kurebayashi ◽  
Kei Saito ◽  
Masami Kodama ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Heart Diseases ◽  
Atomic Level ◽  
Structural Basis ◽  
Ion Binding ◽  
Release Channel ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Cardiac Ryanodine Receptor

AbstractCardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations have been implicated in severe arrhythmogenic heart diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remain unknown. Here, we combined high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated that interactions close to the channel pore are important for stabilizing the channel in the closed state and those in the surrounding regions are essential for channel opening. Our results reveal mechanisms underlying channel opening upon Ca2+ binding and alterations by pathogenic mutations of RyR2 at the atomic level.One Sentence SummaryKey movements and interactions in RyR2 during cardiac Ca2+ channel opening are clarified at the atomic level.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

scholarly journals Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sensen Zhang ◽  
Baolei Yuan ◽  
Jordy Homing Lam ◽  
Jun Zhou ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Md Simulation ◽  
Potential Role ◽  
Md Simulations ◽  
Full Length ◽  
Inflammasome Activation ◽  
Simulation Studies ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Atp Efflux

AbstractPannexin1 (PANX1) is a large-pore ATP efflux channel with a broad distribution, which allows the exchange of molecules and ions smaller than 1 kDa between the cytoplasm and extracellular space. In this study, we show that in human macrophages PANX1 expression is upregulated by diverse stimuli that promote pyroptosis, which is reminiscent of the previously reported lipopolysaccharide-induced upregulation of PANX1 during inflammasome activation. To further elucidate the function of PANX1, we propose the full-length human Pannexin1 (hPANX1) model through cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulation studies, establishing hPANX1 as a homo-heptamer and revealing that both the N-termini and C-termini protrude deeply into the channel pore funnel. MD simulations also elucidate key energetic features governing the channel that lay a foundation to understand the channel gating mechanism. Structural analyses, functional characterizations, and computational studies support the current hPANX1-MD model, suggesting the potential role of hPANX1 in pyroptosis during immune responses.

scholarly journals Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

2021 ◽  
Vol 7 (2) ◽  
pp. eabd4413
Author(s):  
Jung-Hoon Lee ◽  
Daniel Bollschweiler ◽  
Tillman Schäfer ◽  
Robert Huber
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Active Sites ◽  
Chromatin Modification ◽  
Structural Basis ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts ◽  
Lysine Residues ◽  
Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

scholarly journals Brief structural insight into the allosteric gating mechanism of BK (Slo1) channel

2019 ◽  
Vol 97 (6) ◽  
pp. 498-502
Author(s):  
János Almássy ◽  
Péter P. Nánási
Keyword(s):  
Quaternary Structure ◽  
Short Review ◽  
Bk Channel ◽  
The Past ◽  
Gating Model ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Electrophysiological Measurements ◽  
Structural Insight ◽  
Insight Into

The big conductance Ca2+-dependent K+ channel, also known as BK, MaxiK, Slo1, or KCa1.1, is a ligand- and voltage-gated K+ channel. Although structure-function studies of the past decades, involving mutagenesis and electrophysiological measurements, revealed fine details of the mechanism of BK channel gating, the exact molecular details remained unknown until the quaternary structure of the protein has been solved at a resolution of 3.5 Å using cryo-electron microscopy. In this short review, we are going to summarize these results and interpret the gating model of the BK channel in the light of the recent structural results.

scholarly journals Structural insights into the activation of human calcium-sensing receptor

2021 ◽  
Author(s):  
Xiaochen Chen ◽  
Lu Wang ◽  
Zhanyu Ding ◽  
Qianqian Cui ◽  
Li Han ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Transmembrane Domains ◽  
Structural Basis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Cryo Electron Microscopy ◽  
Activation Mechanisms ◽  
G Protein Coupled ◽  
Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

scholarly journals Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

2021 ◽  
Author(s):  
Toby S Turney ◽  
Vivian Li ◽  
Stephen G Brohawn
Keyword(s):  
Conformational Changes ◽  
Pancreatic Beta Cells ◽  
Low Ph ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Lipid Nanodiscs ◽  
Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

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