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 The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase

2019 ◽  
Vol 476 (5) ◽  
pp. 783-794 ◽  
Author(s):  
Lisa Theorin ◽  
Kristina Faxén ◽  
Danny Mollerup Sørensen ◽  
Rebekka Migotti ◽  
Gunnar Dittmar ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Substrate Recognition ◽  
Hydrolytic Activity ◽  
Head Group ◽  
Structural Element ◽  
Type Iv ◽  
Acyl Chains ◽  
Lipid Head Group ◽  
Phospholipid Transport ◽  
P Type

Abstract Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2–ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2–ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.

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 Structural Effects of Cation Binding to DPPC Monolayers

2020 ◽  
Author(s):  
Matti Javanainen ◽  
Wei Hua ◽  
Ondrej Tichacek ◽  
Pauline Delcroix ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Radical Change ◽  
Binding Mode ◽  
Cation Binding ◽  
Head Group ◽  
Binding Modes ◽  
Sum Frequency ◽  
Area Per Lipid ◽  
Simulation And Experiment ◽  
Lipid Head Group ◽  
Dynamics Simulations

Ions at the two sides of the plasma membrane maintain the transmembrane potential, participate in signaling, and affect the properties of the membrane itself. The extracellular leaflet is particularly enriched in phosphatidylcholine lipids an under the influence of Na+, Ca2+, and Cl− ions. In this work, we combined molecular dynamics simulations performed using state-of-the-art models with vibrational sum frequency generation (VSFG) spectroscopy to study the effects of these key ions on the structure of dipalmitoylphosphatidylcholine. We used lipid monolayers as a proxy for membranes, as this approach enabled a direct comparison between simulation and experiment. We find that the effects of Na+ are minor. Ca2+, on the other hand, strongly affects the lipid head group conformations and induces a tighter packing of lipids, thus promoting the liquid condensed phase. It does so by binding to both the phosphate and carbonyl oxygens via direct and water-mediated binding modes, the ratios of which depend on the monolayer packing. Clustering analysis performed on simulation data revealed that changes in area per lipid or CaCl2 concentration both affect the head group conformations, yet their effects are anti-correlated. Cations at the monolayer surface also attract Cl−, which at large CaCl2 concentrations penetrates deep to the monolayer. This phenomenon coincides with a radical change in the VSFG spectra of the phosphate group, thus indicating the emergence of a new binding mode.

scholarly journals Cryo-EM structures capture the transport cycle of the P4-ATPase flippase

Science ◽  
2019 ◽  
Vol 365 (6458) ◽  
pp. 1149-1155 ◽  
Author(s):  
Masahiro Hiraizumi ◽  
Keitaro Yamashita ◽  
Tomohiro Nishizawa ◽  
Osamu Nureki
Keyword(s):  
Membrane Trafficking ◽  
Acyl Chain ◽  
Phosphorylation Site ◽  
Head Group ◽  
Transmembrane Helices ◽  
Type Iv ◽  
Adenosine Triphosphatases ◽  
Lipid Environment ◽  
P Type ◽  
Phosphorylation Domain

In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryo–electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.

scholarly journals Structural Effects of Cation Binding to DPPC Monolayers

2020 ◽  
Author(s):  
Matti Javanainen ◽  
Wei Hua ◽  
Ondrej Tichacek ◽  
Pauline Delcroix ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Radical Change ◽  
Binding Mode ◽  
Cation Binding ◽  
Head Group ◽  
Binding Modes ◽  
Sum Frequency ◽  
Area Per Lipid ◽  
Simulation And Experiment ◽  
Lipid Head Group ◽  
Dynamics Simulations

Ions at the two sides of the plasma membrane maintain the transmembrane potential, participate in signaling, and affect the properties of the membrane itself. The extracellular leaflet is particularly enriched in phosphatidylcholine lipids an under the influence of Na+, Ca2+, and Cl− ions. In this work, we combined molecular dynamics simulations performed using state-of-the-art models with vibrational sum frequency generation (VSFG) spectroscopy to study the effects of these key ions on the structure of dipalmitoylphosphatidylcholine. We used lipid monolayers as a proxy for membranes, as this approach enabled a direct comparison between simulation and experiment. We find that the effects of Na+ are minor. Ca2+, on the other hand, strongly affects the lipid head group conformations and induces a tighter packing of lipids, thus promoting the liquid condensed phase. It does so by binding to both the phosphate and carbonyl oxygens via direct and water-mediated binding modes, the ratios of which depend on the monolayer packing. Clustering analysis performed on simulation data revealed that changes in area per lipid or CaCl2 concentration both affect the head group conformations, yet their effects are anti-correlated. Cations at the monolayer surface also attract Cl−, which at large CaCl2 concentrations penetrates deep to the monolayer. This phenomenon coincides with a radical change in the VSFG spectra of the phosphate group, thus indicating the emergence of a new binding mode.

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.

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.

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

eLife ◽  
2019 ◽  
Vol 8 ◽  
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

The 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 the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold—one of the highest cation gradients known in mammalian tissue—across the membrane.

scholarly journals ATP2, the essential P4-ATPase of malaria parasites, catalyzes lipid-dependent ATP hydrolysis in complex with a Cdc50 β-subunit

2020 ◽  
Author(s):  
Anaïs Lamy ◽  
Ewerton Macarini-Bruzaferro ◽  
Alex Perálvarez-Marín ◽  
Marc le Maire ◽  
José Luis Vázquez-Ibar
Keyword(s):  
Antimalarial Drug ◽  
Drug Target ◽  
Malaria Parasite ◽  
Atp Hydrolysis ◽  
Head Groups ◽  
Phospholipid Distribution ◽  
Phosphatidylinositol 4 ◽  
P Type ◽  
Efficient Mechanisms

ABSTRACTEfficient mechanisms of lipid transport are indispensable for the Plasmodium malaria parasite along the different stages of its intracellular life-cycle. Gene targeting approaches have recently revealed the irreplaceable role of the Plasmodium-encoded type 4 P-type ATPases (P4-ATPases or lipid flippases), ATP2, together with its potential involvement as antimalarial drug target. In eukaryotic membranes, P4-ATPases assure their asymmetric phospholipid distribution by translocating phospholipids from the outer to the inner leaflet. As ATP2 is a yet putative transporter, in this work we have used a recombinantly-produced P. chabaudi ATP2, PcATP2, to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 heterodimerizes with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A, indispensable partners for most P4-ATPases. Moreover, the purified PcATP2/PcCdc50B complex catalyses ATP hydrolysis in the presence of phospholipids containing either phosphatidylserine, phosphatidylethanolamine or phosphatidylcholine head groups, and that this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work provides the first study of the function and quaternary organization of ATP2, a promising antimalarial drug target candidate.

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