scholarly journals What ATP binding does to the Ca2+pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2+

2020 ◽  
Vol 117 (31) ◽  
pp. 18448-18458 ◽  
Author(s):  
Yoshiki Kabashima ◽  
Haruo Ogawa ◽  
Rie Nakajima ◽  
Chikashi Toyoshima
Keyword(s):  
Crystal Structure ◽  
Cardiac Muscle ◽  
Binding Sites ◽  
Phosphorylation Site ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Physiological Conditions ◽  
High Affinity ◽  
Ph Values ◽  
P Domain

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP

1986 ◽  
Vol 64 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Denis LeBel ◽  
Marlyne Beattie
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Photoaffinity Labeling ◽  
Competitive Inhibitor ◽  
Atp Binding ◽  
Zymogen Granule ◽  
Atp Diphosphohydrolase ◽  
High Affinity ◽  
Granule Membrane ◽  
Triton X

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

scholarly journals Comparative modeling of the H4-H5 loop of the α2-isoform of Na+/K+-ATPase α-subunit in the E1 conformation

2007 ◽  
pp. S143-S151
Author(s):  
G Tejral ◽  
L Koláčná ◽  
A Kotyk ◽  
E Amler
Keyword(s):  
Binding Site ◽  
Phosphorylation Site ◽  
Comparative Modeling ◽  
Atp Binding ◽  
Beta Sheet ◽  
Alpha Helices ◽  
Α Subunit ◽  
Atp Binding Site ◽  
P Domain ◽  
Alpha Carbon

Restraint-based comparative modeling was used for calculation and visualization of the H4-H5-loop of Na+/K+-ATPase from mouse brain (Mus musculus, adult male brain, alpha2-isoform) between the amino acid residues Cys 336 and Arg 758 in the E1 conformation The structure consists of two well separated parts. The N-domain is formed by a seven-stranded antiparallel beta-sheet with two additional beta-strands and five alpha-helices sandwiching it, the P-domain is composed of a typical Rossman fold. The ATP-binding site was found on the N-domain to be identical in both alpha2- and alpha1-isoforms. The phosphorylation Asp 369 residue was found in the central part of the P-domain, located at the C-terminal end of the central beta-sheet. The distance between the alpha-carbon of Phe 475 at the ATP-binding site and the alpha-carbon of Asp 369 at the phosphorylation site is 3.22 nm. A hydrogen bond between the oxygen atom of Asp 369 and the nitrogen atom of Lys 690 was clearly detected and assumed to play a key role in maintaining the proper structure of the phosphorylaton site in E1 conformation.

scholarly journals Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

2017 ◽  
Author(s):  
Gracian Tejral ◽  
Bruno Sopko ◽  
Alois Necas ◽  
Wilhelm Schoner ◽  
Evzen Amler
Keyword(s):  
Molecular Modeling ◽  
Computer Modelling ◽  
Phosphorylation Site ◽  
Atp Binding ◽  
Cytoplasmic Loop ◽  
Α Subunit ◽  
Closed Conformation ◽  
Open Conformation ◽  
P Domain ◽  
P Type

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, 3-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information how the transfer of the γ‑phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of 2 ATP binding sites in the open conformation, the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation” -- characterized by a hydrogen bond between Arg543 and Asp611 -- triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

Glutamate receptor binding to cat central nervous system membranes

1980 ◽  
Vol 58 (7) ◽  
pp. 534-538 ◽  
Author(s):  
Richard A. Head ◽  
Godfrey Tunnicliff ◽  
G. Keith Matheson
Keyword(s):  
Spinal Cord ◽  
Binding Sites ◽  
Cervical Spinal Cord ◽  
Specific Binding ◽  
Amygdaloid Complex ◽  
Sodium Ions ◽  
Physiological Conditions ◽  
High Affinity ◽  
Glutamate Binding ◽  
Structural Analogues

L-[3H]Glutamate exhibited specific binding to fresh membranes of cat CNS under physiological conditions of pH and temperature. This binding occurred in the absence of sodium ions. Kinetic analysis of the data for cerebellum suggested the presence of two distinct binding sites: a high-affinity process (Kd = 0.33 μM) with a capacity of 15 pmol/mg protein and a low-affinity process (Kd = 1.8 μM) which had a capacity of 65 pmol/mg protein. Several structural analogues of glutamic acid were able to appreciably inhibit the binding of [3H]glutamate. The distribution of glutamate binding between 12 regions of the CNS was measured. The amygdaloid complex exhibited the highest binding followed by hippocampus > hypothalamus ≡ visual cortex ≡ thalamus ≡ caudate nucleus > olfactory bulb ≡ tectum ≡ cerebellum > dorsal pons ≡ medulla > cervical spinal cord. These findings are consistent with the binding of [3H]glutamate being to its receptor.

scholarly journals Structural basis for murine norovirus engagement of bile acids and the CD300lf receptor

2018 ◽  
Vol 115 (39) ◽  
pp. E9201-E9210 ◽  
Author(s):  
Christopher A. Nelson ◽  
Craig B. Wilen ◽  
Ya-Nan Dai ◽  
Robert C. Orchard ◽  
Arthur S. Kim ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bile Acid ◽  
Bile Acids ◽  
Binding Sites ◽  
Structural Basis ◽  
Interface Residue ◽  
Cellular Receptor ◽  
Murine Norovirus ◽  
Bile Acid Binding ◽  
P Domain

Murine norovirus (MNoV) is closely related to human norovirus (HNoV), an infectious agent responsible for acute gastroenteritis worldwide. Here we report the X-ray crystal structure of the dimeric MNoV VP1 protruding (P) domain in complex with its cellular receptor CD300lf. CD300lf binds the P domain with a 2:2 stoichiometry, engaging a cleft between the AB and DE loops of the P2 subdomain at a site that overlaps the epitopes of neutralizing antibodies. We also identify that bile acids are cofactors enhancing MNoV cell-binding and infectivity. Structures of CD300lf–P domain in complex with glycochenodeoxycholic acid (GCDCA) and lithocholic acid (LCA) reveal two bile acid binding sites at the P domain dimer interface distant from receptor binding sites. The structural determinants for receptor and bile acid binding are supported by numerous biophysical assays utilizing interface residue mutations. We find that the monomeric affinity of CD300lf for the P domain is low and is divalent cation dependent. We have also determined the crystal structure of CD300lf in complex with phosphocholine, revealing that MNoV engages its receptor in a manner mimicking host ligands including similar metal coordination. Docking of the cocomplex structures onto a cryo-EM–derived model of MNoV suggests that each virion can make multiple CD300lf engagements, and thus, infection may be driven by the avidity of cell surface clustered CD300lf. These studies identify multiple potential modulators of norovirus infection that may act to regulate the interaction between the viral capsid P domain and its cognate cellular receptor.

scholarly journals Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

2017 ◽  
Author(s):  
Gracian Tejral ◽  
Bruno Sopko ◽  
Alois Necas ◽  
Wilhelm Schoner ◽  
Evzen Amler
Keyword(s):  
Molecular Modeling ◽  
Computer Modelling ◽  
Phosphorylation Site ◽  
Atp Binding ◽  
Cytoplasmic Loop ◽  
Α Subunit ◽  
Closed Conformation ◽  
Open Conformation ◽  
P Domain ◽  
P Type

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, 3-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information how the transfer of the γ‑phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of 2 ATP binding sites in the open conformation, the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation” -- characterized by a hydrogen bond between Arg543 and Asp611 -- triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

scholarly journals Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3087 ◽  
Author(s):  
Gracian Tejral ◽  
Bruno Sopko ◽  
Alois Necas ◽  
Wilhelm Schoner ◽  
Evzen Amler
Keyword(s):  
Molecular Modeling ◽  
Computer Modelling ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Atp Binding ◽  
Cytoplasmic Loop ◽  
Closed Conformation ◽  
Open Conformation ◽  
P Domain ◽  
P Type

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+and K+transport through cellular membranes leads transiently to a phosphorylation of its catalyticalα-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of theγ-phosphate group of ATP to the Asp369is achieved, analogous molecular modeling of the M4–M5loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338and Ile760of theα2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475in the N-domain, the other one close to Asp369in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

What the structure of a calcium pump tells us about its mechanism

2001 ◽  
Vol 356 (3) ◽  
pp. 665-683 ◽  
Author(s):  
Anthony G. LEE ◽  
J. Malcolm EAST
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Calcium Pump ◽  
Transmembrane Region ◽  
Site Model ◽  
High Affinity ◽  
Cytoplasmic Surface ◽  
Α Helix ◽  
Access Pathway ◽  
Phosphorylation Domain

The report of the crystal structure of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca2+-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647–655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca2+ binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane α-helix M1, with a Ca2+ ion passing through the first site to reach the second site. The Ca2+-ATPase also contains a pair of binding sites for Ca2+ that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca2+ ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca2+ ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca2+ ions must involve a significant change in the packing of the transmembrane α-helices. The link between the phosphorylation domain and the pair of high-affinity Ca2+ binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane α-helices M6 and M7.

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