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.

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

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 Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

2014 ◽  
Vol 70 (6) ◽  
pp. 1622-1630 ◽  
Author(s):  
Sina Möhlmann ◽  
Rebecca Mathew ◽  
Piotr Neumann ◽  
Andreas Schmitt ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Binding Activity ◽  
Mrna Splicing ◽  
Structural Basis ◽  
Atp Binding ◽  
Helicase Domain ◽  
Crucial Step ◽  
Closed Conformation ◽  
Dead Box ◽  
Open Conformation ◽  
U1 Snrnp

The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

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 Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Diane T. Takahashi ◽  
Danièle Gadelle ◽  
Keli Agama ◽  
Evgeny Kiselev ◽  
Hongliang Zhang ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Conformational Transition ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Torsional Stress ◽  
Enzyme Dynamics ◽  
Dna Molecule ◽  
Closed Conformation ◽  
Open Conformation

AbstractEukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

scholarly journals The π-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase

2009 ◽  
pp. 583-589
Author(s):  
G Tejral ◽  
L Koláčná ◽  
W Schoner ◽  
E Amler
Keyword(s):  
Molecular Modeling ◽  
Atpase Activity ◽  
Binding Site ◽  
Conformational Change ◽  
Phosphorylation Site ◽  
Hinge Region ◽  
Atp Binding Site ◽  
Helix Formation ◽  
Key Factor ◽  
P Domain

Molecular modeling of the H4-H5-loop of the α2 isoform of Na+/K+- ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short π-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7° and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+- ATPase activity.

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.

scholarly journals Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Komal Soni ◽  
Georg Kempf ◽  
Karen Manalastas-Cantos ◽  
Astrid Hendricks ◽  
Dirk Flemming ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Sites ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Closed Conformation ◽  
Open Conformation ◽  
Structural Database ◽  
Alu Rna ◽  
Species Specific ◽  
Protozoan Species

AbstractThe eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly ‘closed’ conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an ‘open’ Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.

scholarly journals Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingmin Yuan ◽  
Zongyang Lv ◽  
Melanie J. Adams ◽  
Shaun K. Olsen
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Molecular Mechanisms ◽  
Conformational State ◽  
Biochemical Data ◽  
Conformational States ◽  
Product Release ◽  
Closed Conformation ◽  
Open Conformation ◽  
Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

scholarly journals Three-Dimensional Structures of Carbohydrates and Where to Find Them

2020 ◽  
Vol 21 (20) ◽  
pp. 7702 ◽  
Author(s):  
Sofya I. Scherbinina ◽  
Philip V. Toukach
Keyword(s):  
Experimental Data ◽  
Molecular Modeling ◽  
Computational Methods ◽  
Three Dimensional ◽  
Structural Diversity ◽  
Structural Data ◽  
Structural Features ◽  
Data Validation ◽  
Data Generation ◽  
Efficient Treatment

Analysis and systematization of accumulated data on carbohydrate structural diversity is a subject of great interest for structural glycobiology. Despite being a challenging task, development of computational methods for efficient treatment and management of spatial (3D) structural features of carbohydrates breaks new ground in modern glycoscience. This review is dedicated to approaches of chemo- and glyco-informatics towards 3D structural data generation, deposition and processing in regard to carbohydrates and their derivatives. Databases, molecular modeling and experimental data validation services, and structure visualization facilities developed for last five years are reviewed.

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