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.

Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin

1998 ◽  
Vol 4 (S2) ◽  
pp. 462-463
Author(s):  
P. Zhang ◽  
C. Toyoshima ◽  
K. Yonekura ◽  
G. Inesi ◽  
M. Green ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Large Family ◽  
Calcium Pump ◽  
Dimensional Structure ◽  
Transmembrane Helices ◽  
Site Mutagenesis ◽  
P Type

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

scholarly journals Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ye Zhou ◽  
Can Cao ◽  
Lingli He ◽  
Xianping Wang ◽  
Xuejun Cai Zhang
Keyword(s):  
Crystal Structure ◽  
Dopamine Receptor ◽  
Binding Pocket ◽  
Structural Features ◽  
Transmembrane Helices ◽  
Subtype Selectivity ◽  
Selective Ligand ◽  
Neurological Processes ◽  
Subtype Selective ◽  
Dopamine Receptor D4

Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.

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.

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 Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X

2019 ◽  
Vol 116 (15) ◽  
pp. 7288-7297 ◽  
Author(s):  
Prajwal Paudel ◽  
Qi Zhang ◽  
Charles Leung ◽  
Harrison C. Greenberg ◽  
Yusong Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Substrate Recognition ◽  
Structural Features ◽  
Catalytic Triad ◽  
Specific Substrate ◽  
Structure Model ◽  
Cellular Processes ◽  
Structure And Activity

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the β-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

scholarly journals Structures of a P4-ATPase lipid flippase in lipid bilayers

2020 ◽  
Author(s):  
Yilin He ◽  
Jinkun Xu ◽  
Xiaofei Wu ◽  
Long Li
Keyword(s):  
Lipid Bilayers ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Transmembrane Segment ◽  
Lipid Asymmetry ◽  
Chaetomium Thermophilum ◽  
Intermediate States ◽  
P Type ◽  
Lipid Nanodiscs ◽  
Lipid Flippase

AbstractType 4 P-type ATPases (P4-ATPases) are a group of key enzymes maintaining lipid asymmetry of eukaryotic membranes. Phospholipids are actively and selectively flipped by P4-ATPases from the exoplasmic leaflet to the cytoplasmic leaflet. How lipid flipping is coupled with ATP-hydrolysis by P4-ATPases is poorly understood. Here, we report the electron cryo-microscopy structures of a P4-ATPase, Dnf1-Cdc50 from Chaetomium thermophilum, which had been reconstituted into lipid nanodiscs and captured in two transport intermediate states. The structures reveal that transmembrane segment 1 of Dnf1 becomes highly flexible during lipid transport. The local lipid bilayers are distorted to facilitate the entry of the phospholipid substrates from the exoplasmic leaflet to a cross-membrane groove. During transport, the lipid substrates are relayed through four binding sites in the groove which constantly shields the lipid polar heads away from the hydrophobic environment of the membranes.

scholarly journals Effects of phosphatidylethanolamines on the activity of the Ca2+-ATPase of sarcoplasmic reticulum

1996 ◽  
Vol 320 (1) ◽  
pp. 309-314 ◽  
Author(s):  
Anthony P STARLING ◽  
Kate A DALTON ◽  
J. Malcolm EAST ◽  
Susan OLIVER ◽  
Anthony G LEE
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Equilibrium Constant ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Egg Yolk ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Steady State Rate ◽  
State Rate

ATPase activities for the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted into dioleoylphosphatidylethanolamine [di(C18:1)PE] are, at temperatures higher than 20 °C, lower than in dioleoylphosphatidylcholine [di(C18:1)PC], whereas in egg yolk phosphatidylethanolamine the activities are the same as in di(C18:1)PC up to 25 °C, suggesting that low ATPase activities occur when the phosphatidylethanolamine species is in the hexagonal HII phase. ATPase activities measured in mixtures of di(C18:1)PC and di(C18:1)PE do not change with changing di(C18:1)PE content up to 80%. It is concluded that curvature frustration in bilayers containing di(C18:1)PE has no effect on ATPase activity. The rates of phosphorylation and of Ca2+ transport are identical for the native ATPase and for the ATPase in di(C18:1)PE. Dephosphorylation of the phosphorylated ATPase in di(C18:1)PE at 25 °C is, however, slower than for the native ATPase, explaining the lower steady-state rate of ATP hydrolysis; in egg yolk phosphatidylethanolamine at 25 °C the rate of dephosphorylation is equal to that for the unreconstituted ATPase. Phosphorylation of the ATPase by Pi in the absence of Ca2+ is unaffected by reconstitution in di(C18:1)PE. The stoichiometry of Ca2+ binding to the ATPase is also unaltered. Studies of the effect of di(C18:1)PE on the fluorescence intensity of the ATPase labelled with 7-chloro-4-nitro-2,1,3-benzoxadiazole are consistent with an increase in the E1/E2 equilibrium constant, where E1 is the conformation of the ATPase with two high-affinity binding sites for Ca2+ exposed to the cytoplasm, and E2 is a conformation unable to bind cytoplasmic Ca2+. A slight increase in affinity for Ca2+ can be attributed to the observed increase in the E1/E2 equilibrium constant.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

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.

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