scholarly journals Comparative Analyses of theβ-Tubulin Gene and Molecular Modeling Reveal Molecular Insight into the Colchicine Resistance in Kinetoplastids Organisms

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Luis Luis ◽  
María Luisa Serrano ◽  
Mariana Hidalgo ◽  
Alexis Mendoza-León
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Differential Susceptibility ◽  
Binding Pocket ◽  
Crystallographic Structure ◽  
Tubulin Gene ◽  
Colchicine Binding Site ◽  
Colchicine Resistance ◽  
Leishmania Spp

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such asLeishmania spp. andTrypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site ofLeishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced theβ-tubulin gene ofLeishmania (Viannia) guyanensisand established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on theLeishmania  β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in theβ-tubulin sequence of kinetoplastid organisms such asTrypanosoma cruzi,T. brucei, andT. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of anα-helix structure and displacement to the inside of the pocket of oneβ-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

G-actin conformational change and polymerization induced by paraquat

1998 ◽  
Vol 76 (4) ◽  
pp. 583-591 ◽  
Author(s):  
Isabella DalleDonne ◽  
Aldo Milzani ◽  
Roberto Colombo
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Cell Injury ◽  
Atp Hydrolysis ◽  
Protein Composition ◽  
Dnase I ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Actin Assembly

Paraquat (1,1´-dimethyl-4,4´-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.Key words: actin, paraquat, subdomain 2, DNase I, ATP hydrolysis.

scholarly journals An allosteric transition trapped in an intermediate state of a new kinesin–inhibitor complex

2009 ◽  
Vol 425 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Hung Yi Kristal Kaan ◽  
Venkatasubramanian Ulaganathan ◽  
David D. Hackney ◽  
Frank Kozielski
Keyword(s):  
Conformational Changes ◽  
Intermediate State ◽  
Drug Target ◽  
Structural Changes ◽  
Binding Pocket ◽  
Bound State ◽  
Inhibitor Binding ◽  
Drug Induced ◽  
Inhibitor Complex ◽  
Kinesin Eg5

Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with seven inhibitors already in clinical trials. In the present paper, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor STLC (S-trityl-L-cysteine) to 2.0 Å (1 Å=0.1 nm) resolution. The Eg5–STLC complex crystallizes in space group P32 with three molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby loop L5 has swung downwards to close the inhibitor-binding pocket, helix α4 has rotated by approx. 15 ° and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck-linker. This provides structural evidence for the sequence of drug-induced conformational changes.

scholarly journals Conformational dynamics of androgen receptors bound to agonists and antagonists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hyo Jin Gim ◽  
Jiyong Park ◽  
Michael E. Jung ◽  
K. N. Houk
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Structural Information ◽  
Close Contact ◽  
Conformational Dynamics ◽  
Principal Component ◽  
Binding Pocket ◽  
Structural Basis ◽  
Accelerated Molecular Dynamics

AbstractThe androgen receptor (AR) is critical in the progression of prostate cancer (PCa). Small molecule antagonists that bind to the ligand binding domain (LBD) of the AR have been successful in treating PCa. However, the structural basis by which the AR antagonists manifest their therapeutic efficacy remains unclear, due to the lack of detailed structural information of the AR bound to the antagonists. We have performed accelerated molecular dynamics (aMD) simulations of LBDs bound to a set of ligands including a natural substrate (dihydrotestosterone), an agonist (RU59063) and three antagonists (bicalutamide, enzalutamide and apalutamide) as well as in the absence of ligand (apo). We show that the binding of AR antagonists at the substrate binding pocket alter the dynamic fluctuations of H12, thereby disrupting the structural integrity of the agonistic conformation of AR. Two antagonists, enzalutamide and apalutamide, induce considerable structural changes to the agonist conformation of LBD, when bound close to H12 of AR LBD. When the antagonists bind to the pocket with different orientations having close contact with H11, no significant conformational changes were observed, suggesting the AR remains in the functionally activated (agonistic) state. The simulations on a drug resistance mutant F876L bound to enzalutamide demonstrated that the mutation stabilizes the agonistic conformation of AR LBD, which compromises the efficacy of the antagonists. Principal component analysis (PCA) of the structural fluctuations shows that the binding of enzalutamide and apalutamide induce conformational fluctuations in the AR, which are markedly different from those caused by the agonist as well as another antagonist, bicalutamide. These fluctuations could only be observed with the use of aMD.

scholarly journals pH-Induced Conformational Changes inClostridium difficile Toxin B

2000 ◽  
Vol 68 (5) ◽  
pp. 2470-2474 ◽  
Author(s):  
Maen Qa'Dan ◽  
Lea M. Spyres ◽  
Jimmy D. Ballard
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Tryptophan Fluorescence ◽  
Acidic Ph ◽  
Bafilomycin A1 ◽  
Toxin B ◽  
Endosomal Acidification ◽  
Specific Protease ◽  
The Impact

ABSTRACT Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.

scholarly journals Structural Changes in the Cap of Rv0183/mtbMGL Modulate the Shape of the Binding Pocket

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1299
Author(s):  
Christoph Grininger ◽  
Mario Leypold ◽  
Philipp Aschauer ◽  
Tea Pavkov-Keller ◽  
Lina Riegler-Berket ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Treatment Strategies ◽  
Active Phase ◽  
Building Blocks ◽  
Binding Pocket ◽  
Multidrug Resistant ◽  
Free Form ◽  
Closed Conformation ◽  
Substrate Inhibitor

Tuberculosis continues to be a major threat to the human population. Global efforts to eradicate the disease are ongoing but are hampered by the increasing occurrence of multidrug-resistant strains of Mycobacterium tuberculosis. Therefore, the development of new treatment, and the exploration of new druggable targets and treatment strategies, are of high importance. Rv0183/mtbMGL, is a monoacylglycerol lipase of M. tuberculosis and it is involved in providing fatty acids and glycerol as building blocks and as an energy source. Since the lipase is expressed during the dormant and active phase of an infection, Rv0183/mtbMGL is an interesting target for inhibition. In this work, we determined the crystal structures of a surface-entropy reduced variant K74A Rv0183/mtbMGL in its free form and in complex with a substrate mimicking inhibitor. The two structures reveal conformational changes in the cap region that forms a major part of the substrate/inhibitor binding region. We present a completely closed conformation in the free form and semi-closed conformation in the ligand-bound form. These conformations differ from the previously published, completely open conformation of Rv0183/mtbMGL. Thus, this work demonstrates the high conformational plasticity of the cap from open to closed conformations and provides useful insights into changes in the substrate-binding pocket, the target of potential small-molecule inhibitors.

scholarly journals Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthieu P.M.H. Benoit ◽  
Ana B. Asenjo ◽  
Mohammadjavad Paydar ◽  
Sabin Dhakal ◽  
Benjamin H. Kwok ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Bound State ◽  
Structural Basis ◽  
Coupling Mechanism ◽  
Developmental Defects ◽  
Microtubule Binding

AbstractKIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7–3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

scholarly journals Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

2019 ◽  
Vol 295 (6) ◽  
pp. 1551-1564 ◽  
Author(s):  
Kelly E. Du Pont ◽  
Russell B. Davidson ◽  
Martin McCullagh ◽  
Brian J. Geiss
Keyword(s):  
Molecular Dynamics ◽  
Structural Changes ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Binding Pocket ◽  
Energy Transduction ◽  
Helicase Domain ◽  
Genome Replication ◽  
Turnover Rates

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

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