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 Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I

Nature ◽  
1994 ◽  
Vol 367 (6459) ◽  
pp. 138-146 ◽  
Author(s):  
Christopher D. Lima ◽  
James C. Wang ◽  
Alfonso Mondragón
Keyword(s):  
Topoisomerase I ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Three Dimensional Structure ◽  
E Coli ◽  
Terminal Fragment

scholarly journals Two Functional States of the CD11b A-Domain: Correlations with Key Features of Two Mn2+-complexed Crystal Structures

1998 ◽  
Vol 143 (6) ◽  
pp. 1523-1534 ◽  
Author(s):  
Rui Li ◽  
Philippe Rieu ◽  
Diana L. Griffith ◽  
David Scott ◽  
M. Amin Arnaout
Keyword(s):  
Active Form ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Side Chain ◽  
Wild Type ◽  
Solvent Exposure ◽  
Open Conformation ◽  
Functional States ◽  
Open Versus ◽  
Metal Ligand

In the presence of bound Mn2+, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the “open” conformation previously described only for a crystalline Mg2+ complex. The open conformation is distinguished from the “closed” form by the solvent exposure of F302, a direct T209–Mn2+ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an “active” state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.

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.

Supercoils in human DNA

1975 ◽  
Vol 19 (2) ◽  
pp. 261-279
Author(s):  
P.R. Cook ◽  
I.A. Brazell
Keyword(s):  
Gamma Rays ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Hydrodynamic Properties ◽  
Dna Molecule ◽  
Double Stranded Dna ◽  
Human Dna ◽  
Nuclear Structures ◽  
Intercalating Agents ◽  
Characteristic Manner

The three-dimensional structure of a double-stranded DNA molecule may be described by distinguishing the helical turns of the DNA duplex from any superhelical turns that might be superimposed upon the duplex turns. There are characteristic changes in the hydrodynamic properties of superhelical DNA molecules when they interact with intercalating agents. The hydrodynamic properties of nuclear structures released by gently lysing human cells are changed by intercalating agents in this characteristic manner. The characteristic changes are abolished by irradiating the cells with gamma-rays but may be restored by incubating the cells at 37 degrees C after irradiation. These results are interpreted as showing that human DNA is supercoiled. A model for the structure of the chromosome is suggested.

scholarly journals The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

2004 ◽  
Vol 380 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Young Sam SEO ◽  
Ahrim YOO ◽  
Jinwon JUNG ◽  
Soon-Kee SUNG ◽  
Dae Ryook YANG ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Comparative Modelling

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight α-helices, 12 β-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called ‘2-His-1-carboxylate facial triad’. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

scholarly journals Probing the Determinants of Coenzyme Specificity in Ferredoxin-NADP+Reductase by Site-directed Mutagenesis

2001 ◽  
Vol 276 (15) ◽  
pp. 11902-11912 ◽  
Author(s):  
Milagros Medina ◽  
Alejandra Luquita ◽  
Jesús Tejero ◽  
Juan Hermoso ◽  
Tomás Mayoral ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Structure Comparison ◽  
Coenzyme Specificity ◽  
Related Family ◽  
Secondary Interactions ◽  
Main Determinants

On the basis of sequence and three-dimensional structure comparison betweenAnabaenaPCC7119 ferredoxin-NADP+reductase (FNR) and other reductases from its structurally related family that bind either NADP+/H or NAD+/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2′-phosphate of NADP+/H inAnabaenaFNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP+/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions inAnabaenaFNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2′-phosphate NADP+/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261–268 inAnabaenaFNR must be one of these regions.

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