scholarly journals The SERCA residue Glu340 mediates inter-domain communication that guides Ca2+ transport

2020 ◽  
Author(s):  
Maxwell M. G. Geurts ◽  
Johannes D. Clausen ◽  
Bertrand Arnou ◽  
Cedric Montigny ◽  
Guillaume Lenoir ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Transmembrane Helices ◽  
Tight Coupling ◽  
Hydrogen Bonding Pattern ◽  
Plasmic Reticulum ◽  
Cytosolic Domains ◽  
Dynamics Simulations ◽  
P Type ◽  
Intramolecular Communication

AbstractThe sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the SR/ER lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+ binding sites and of the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of five out of SERCA’s ten transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analogue reveals a rotated headpiece, altered connectivity between the cytosolic domains and altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in inter-domain communication between the headpiece and the Ca2+-binding transmembrane region.

scholarly journals The SERCA residue Glu340 mediates interdomain communication that guides Ca2+transport

2020 ◽  
Vol 117 (49) ◽  
pp. 31114-31122
Author(s):  
Maxwell M. G. Geurts ◽  
Johannes D. Clausen ◽  
Bertrand Arnou ◽  
Cédric Montigny ◽  
Guillaume Lenoir ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Transmembrane Helices ◽  
Tight Coupling ◽  
Hydrogen Bonding Pattern ◽  
Plasmic Reticulum ◽  
Cytosolic Domains ◽  
Dynamics Simulations ◽  
P Type ◽  
Intramolecular Communication ◽  
Interdomain Communication

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA’s 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5505 ◽  
Author(s):  
Alexander Krah ◽  
Peter J. Bond
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Evolutionary Analysis ◽  
Binding Affinities ◽  
Wild Type ◽  
Dynamics Simulations ◽  
High Binding Affinity ◽  
Atp Synthases

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

2001 ◽  
Vol 356 (3) ◽  
pp. 685-704 ◽  
Author(s):  
Kathleen J. SWEADNER ◽  
Claudia DONNET
Keyword(s):  
Crystal Structure ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Structural Features ◽  
Cleavage Sites ◽  
Transmembrane Helices ◽  
Transmembrane Topology ◽  
Key Features ◽  
P Type

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 Å = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca2+-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca2+-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

scholarly journals A single K+-binding site in the crystal structure of the gastric proton pump

2019 ◽  
Author(s):  
Kenta Yamamoto ◽  
Vikas Dubey ◽  
Katsumasa Irie ◽  
Hanayo Nakanishi ◽  
Himanshu Khandelia ◽  
...  
Keyword(s):  
Binding Site ◽  
Proton Pump ◽  
Atp Hydrolysis ◽  
Energy Requirement ◽  
Cation Binding ◽  
Structural Basis ◽  
Cation Binding Site ◽  
Gastric Proton Pump ◽  
Dynamics Simulations ◽  
P Type

AbstractThe gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold – the highest cation gradient known in any mammalian tissue – across the membrane.

The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump

2010 ◽  
Vol 43 (4) ◽  
pp. 501-566 ◽  
Author(s):  
Jesper V. Møller ◽  
Claus Olesen ◽  
Anne-Marie L. Winther ◽  
Poul Nissen
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transport Mechanism ◽  
Atp Hydrolysis ◽  
Cation Binding ◽  
Thermodynamic Efficiency ◽  
Lipid Phase ◽  
Conformational States ◽  
Sarcoplasmic Reticulum Membrane ◽  
Cytosolic Domains ◽  
P Type

AbstractThe sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal mucles where it functions as an indispensable component of the excitation–contraction coupling, being at the expense of ATP hydrolysis involved in Ca2+/H+ exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca2+ transporting proteins count Na+, K+-ATPase and H+, K+-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3–3 Å) resolution 3D-crystals of Ca2+-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca2+-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca2+ and H+, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca2+ across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.

What Molecular Dynamics Simulations Can Tell Us About Mechanical Properties of Kinesin and Its Interaction With Tubulin

2007 ◽  
Author(s):  
Iuliana Aprodu ◽  
Alfonso Gautieri ◽  
Franco M. Montevecchi ◽  
Alberto Redaelli ◽  
Monica Soncini
Keyword(s):  
Mechanical Properties ◽  
Molecular Dynamics ◽  
Light Chain ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Metabolic Energy ◽  
Hand Model ◽  
Membrane Bound ◽  
Dynamics Simulations

Kinesin is a processive molecular motor found in various cells including neurons, that transports membrane-bound vesicles and organelles along the microtubule. Kinesin typically consists of three distinct domains: two large globular heads that attach to the microtubule, a central coiled region, and a light-chain that attaches to the cellular cargo. The metabolic energy that drives kinesins is provided in the form of ATP. The energy released by ATP hydrolysis is converted into direct movement after kinesin binds strongly to the microtubule. Two mechanisms were proposed to explain the movement of kinesin along microtubules: the “hand over hand” model in which the two heads alternate in the role of leading and the “inchworm” model in which one head always leads.

scholarly journals Computational Studies of Substrate Transport and Specificity in a Phospholipid Flippase

2020 ◽  
Author(s):  
Yong Wang ◽  
Joseph A Lyons ◽  
Milena Timcenko ◽  
Bert L. de Groot ◽  
Poul Nissen ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Head Group ◽  
Outer Leaflet ◽  
Type Iv ◽  
Lipid Head Group ◽  
Dynamics Simulations ◽  
Phospholipid Distribution ◽  
Phospholipid Transport ◽  
P Type ◽  
Insight Into

AbstractType-IV P-type ATPases are lipid flippases which help maintain asymmetric phospholipid distribution in eukaryotic membranes by using ATP hydrolysis to drive unidirectional translocation of phospholipid substrates. Recent Cryo-EM and crystal structures have provided a detailed view of flippases, and we here use molecular dynamics simulations of the yeast flippase Drs2p:Cdc50p in an outward open conformation to study the first steps of phospholipid transport. Our simulations show phospholipid binding to a groove and subsequent movement towards the centre of the membrane, and reveal a preference for phosphatidylserine lipids. We find that the lipid head group stays solvated in the groove while the lipid tails stay in the membrane during the (half) transport event. The flippase also induces deformation and thinning of the outer leaflet. Together, our simulations provide insight into substrate binding to lipid flippases and suggest that multiple sites and steps in the functional cycle contribute to substrate selectivity.

scholarly journals Role of the self-association of β subunits in the oligomeric structure of Na+/K+-ATPase

2002 ◽  
Vol 364 (1) ◽  
pp. 293-299 ◽  
Author(s):  
Alexander V. IVANOV ◽  
Nikolai N. MODYANOV ◽  
Amir ASKARI
Keyword(s):  
Spatial Relations ◽  
Cross Linking ◽  
Transmembrane Helices ◽  
Oligomeric Structure ◽  
Membrane Bound ◽  
Self Association ◽  
Quaternary Structures ◽  
Α Helix ◽  
P Type

The two subunits of Na+/K+-ATPase that are essential for function are α and β. Previous cross-linking studies on the oligomeric structure of the membrane-bound enzyme identified α,β and α,α associations, but only the former and not the latter could be detected after solubilization. To study the possibility of direct β,β association, the purified membrane enzyme and a trypsin-digested enzyme that occludes cations and contains an essentially intact β and fragments of α were subjected to oxidative cross-linking in the presence of Cu2+-phenanthroline. Resolution of products on polyacrylamide gels, N-terminal analysis and reactivity with anti-β antibody showed that, in addition to previously identified products (e.g. α,α and α,β dimers), a β,β dimer, most likely linked through intramembrane Cys44 residues of two chains, is also formed. This dimer was also noted when digitonin-solubilized intact enzyme, and the trypsin-digested enzyme solubilized with digitonin or polyoxyethylene 10-laurylether were subjected to cross-linking, indicating that the detected β,β association was not due to random collisions. In the digested enzyme, K+ but not Na+ enhanced β,β dimer formation. The alternative cross-linking of β-Cys44 to a Cys residue of a transmembrane α-helix was antagonized specifically by K+ or Na+. The findings (i) indicate the role of β,β association in maintaining the minimum oligomeric structure of (α,β)2, (ii) provide further support for conformation-dependent flexibilities of the spatial relations of the transmembrane helices of α and β and (iii) suggest the possibility of significant differences between the quaternary structures of the P-type ATPases that do and do not contain a β subunit.

scholarly journals Cryo-EM structure of the mechanically activated ion channel OSCA1.2

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sebastian Jojoa-Cruz ◽  
Kei Saotome ◽  
Swetha E Murthy ◽  
Che Chun Alex Tsui ◽  
Mark SP Sansom ◽  
...  
Keyword(s):  
Ion Channels ◽  
Turgor Pressure ◽  
Ion Permeation ◽  
Membrane Tension ◽  
Transmembrane Helices ◽  
Channel Conductance ◽  
Cryo Electron Microscopy ◽  
Stress Sensing ◽  
Dynamics Simulations

Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. Here, we elucidate high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. We locate the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels.

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