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 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.

Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

2014 ◽  
Vol 112 (07) ◽  
pp. 53-64 ◽  
Author(s):  
Sven Brandt ◽  
Krystin Krauel ◽  
Kay E. Gottschalk ◽  
Thomas Renné ◽  
Christiane A. Helm ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Cd Spectroscopy ◽  
Platelet Factor 4 ◽  
Drug Induced ◽  
Platelet Factor ◽  
Preclinical Drug Development ◽  
Varying Length ◽  
Endogenous Proteins

SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

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.

Molecular Dissection of the Inhibitor Binding Pocket of Mitotic Kinesin Eg5 Reveals Mutants that Confer Resistance to Antimitotic Agents

2006 ◽  
Vol 360 (2) ◽  
pp. 360-376 ◽  
Author(s):  
Sébastien Brier ◽  
David Lemaire ◽  
Salvatore DeBonis ◽  
Eric Forest ◽  
Frank Kozielski
Keyword(s):  
Binding Pocket ◽  
Molecular Dissection ◽  
Inhibitor Binding ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5

scholarly journals An Induced Fit Conformational Change Underlies the Binding Mechanism of the Heme Transport Proteobacteria-Protein HemS

2006 ◽  
Vol 281 (43) ◽  
pp. 32606-32610 ◽  
Author(s):  
Sabine Schneider ◽  
Katherine H. Sharp ◽  
Paul D. Barker ◽  
Max Paoli
Keyword(s):  
Conformational Changes ◽  
Structural Data ◽  
Binding Pocket ◽  
Bound State ◽  
Uptake System ◽  
Heme Binding ◽  
Binding Mechanism ◽  
Induced Fit ◽  
Heme Transport ◽  
Β Sheet

Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large β-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a “heme-induced fit” binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.

Drug–target identification from total cellular lysate by drug-induced conformational changes

2009 ◽  
Vol 385 (2) ◽  
pp. 314-320 ◽  
Author(s):  
Yoichi Nishiya ◽  
Kenji Shibata ◽  
Seiji Saito ◽  
Keiichi Yano ◽  
Chitose Oneyama ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Target ◽  
Target Identification ◽  
Drug Induced ◽  
Drug Target Identification

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.

Functionalized Homologues and Positional Isomers of Rabbit 15-Lipoxygenase RS75091 Inhibitor

2021 ◽  
Vol 17 ◽  
Author(s):  
Alexander Zhuravlev ◽  
Alexey Golovanov ◽  
Valery Toporkov ◽  
Hartmut Kuhn ◽  
Igor Ivanov
Keyword(s):  
Conformational Changes ◽  
Structural Alignment ◽  
Coupling Reaction ◽  
Structural Data ◽  
Binding Pocket ◽  
Cinnamic Acid Derivative ◽  
Inhibitor Complex ◽  
Sonogashira Coupling Reaction ◽  
Linker Group ◽  
Aliphatic Spacer

Background: RS75091 is a cinnamic acid derivative that has been used for the crystallization of the rabbit ALOX15-inhibitor complex. The atomic coordinates of the resolved ALOX15-inhibitor complex were later used to define the binding sites of other mammalian lipoxygenase orthologs, for which no direct structural data with ligand has been reported so far. Introduction: The putative binding pocket of the human ALOX5 was reconstructed on the basis of its structural alignment with rabbit ALOX15-RS75091 inhibitor. However, considering the possible conformational changes the enzyme may undergo in solution, it remains unclear whether the existing models adequately mirror the architecture of the ALOX5 active site. Methods: In this study, we prepared a series of RS75091 derivatives using a Sonogashira coupling reaction of regioisomeric bromocinnamates with protected acetylenic alcohols and tested their inhibitory properties on rabbit ALOX15 Results: A bulky pentafluorophenyl moiety linked to either ortho- or metha-ethynylcinnamates via aliphatic spacer does not significantly impair the inhibitory properties of RS75091. Conclusion: Hydroxylated 2- and 3-alkynylcinnamates may be suitable candidates for incorporation of an aromatic linker group like tetrafluorophenylazides for photoaffinity labeling assays.

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.

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°.

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