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 Nucleotide-Dependent Conformational Changes in the σ54-Dependent Activator DctD

2003 ◽  
Vol 185 (20) ◽  
pp. 6215-6219 ◽  
Author(s):  
Ying-Kai Wang ◽  
Sungdae Park ◽  
B. Tracy Nixon ◽  
Timothy R. Hoover
Keyword(s):  
Conformational Changes ◽  
Sinorhizobium Meliloti ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Active Form ◽  
Dnase I ◽  
Promoter Complex ◽  
Arginine Finger ◽  
Rna Polymerase Holoenzyme ◽  
Fluorescence Energy

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctDΔ1-142, a truncated and constitutively active form of the σ54-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctDΔ1-142 during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2′(3′)-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctDΔ1-142 and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.

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.

scholarly journals Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

2020 ◽  
Vol 48 (20) ◽  
pp. 11664-11674 ◽  
Author(s):  
Ivana Duic ◽  
Hisashi Tadakuma ◽  
Yoshie Harada ◽  
Ryo Yamaue ◽  
Katashi Deguchi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Antiviral Response ◽  
Fiber Formation ◽  
Viral Rna ◽  
Active Conformation ◽  
Viral Dsrna ◽  
Fiber Assembly ◽  
Signaling Domain

Abstract Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

scholarly journals Nucleosome Remodeling by the Human SWI/SNF Complex Requires Transient Global Disruption of Histone-DNA Interactions

2002 ◽  
Vol 22 (11) ◽  
pp. 3653-3662 ◽  
Author(s):  
Sayura Aoyagi ◽  
Geeta Narlikar ◽  
Chunyang Zheng ◽  
Saïd Sif ◽  
Robert E. Kingston ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Atp Hydrolysis ◽  
Dnase I ◽  
Cross Linking ◽  
Dna Interactions ◽  
Nucleosome Remodeling ◽  
Histone Octamer ◽  
Nucleosomal Dna ◽  
Single Cross ◽  
A Site

ABSTRACT We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.

A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB

2005 ◽  
Vol 33 (5) ◽  
pp. 990-995 ◽  
Author(s):  
J. Zaitseva ◽  
S. Jenewein ◽  
C. Oswald ◽  
T. Jumpertz ◽  
I.B. Holland ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Central Element ◽  
Nucleotide Binding ◽  
Single Step ◽  
Catalytic Cycle ◽  
Type I ◽  
Individual Crystal

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

2007 ◽  
Vol 13 (4) ◽  
pp. 281-290 ◽  
Author(s):  
Petr Novak ◽  
Vladimir Havlicek ◽  
Peter J. Derrick ◽  
Kyle A. Beran ◽  
Sajid Bashir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Cyclotron Resonance ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Cyclotron Resonance ◽  
Ion Cyclotron ◽  
Chemical Cross Linking

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes in proteins induced by ligand binding and to study the effects these changes have on non-covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium-free calmodulin form is the same as in the calcium-loaded calmodulin/melittin complex.

scholarly journals Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

2016 ◽  
Author(s):  
Nathan D. Thomsen ◽  
Michael R. Lawson ◽  
Lea B. Witkowsky ◽  
Song Qu ◽  
James M. Berger
Keyword(s):  
Nucleic Acid ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Ring Closure ◽  
Structural Studies ◽  
Saxs Data ◽  
Structural Methods

ABSTRACTRing-shaped hexameric helicases and translocases support essential DNA, RNA, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS data show that Rho preferentially adopts an open-ring state in solution, and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish the first structural snapshots of substepping events in a hexameric helicase/translocase.SIGNIFICANCEHexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.

scholarly journals Oligomerization of EpsE Coordinates Residues from Multiple Subunits to Facilitate ATPase Activity

2011 ◽  
Vol 286 (12) ◽  
pp. 10378-10386 ◽  
Author(s):  
Marcella Patrick ◽  
Konstantin V. Korotkov ◽  
Wim G. J. Hol ◽  
Maria Sandkvist
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Hydrolytic Enzymes ◽  
Point Mutations ◽  
Nucleotide Binding ◽  
Type Ii ◽  
Arginine Residues

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the Gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg210, Arg225, Arg320, Arg324, Arg336, and Arg369) from adjoining domains and subunits to complete the active site around the bound nucleotide. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.

scholarly journals The role of ATP in the function of human ORC4 protein

2010 ◽  
Vol 75 (3) ◽  
pp. 317-322
Author(s):  
Aleksandra Divac ◽  
Branko Tomic ◽  
Jelena Kusic
Keyword(s):  
Conformational Changes ◽  
Origin Recognition Complex ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Structural Role ◽  
A Subunit ◽  
Dna Substrates ◽  
Origin Recognition

Human ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of ATPases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.

scholarly journals Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus

2013 ◽  
Vol 450 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Richard F. Collins ◽  
Darin Hassan ◽  
Vijaykumar Karuppiah ◽  
Angela Thistlethwaite ◽  
Jeremy P. Derrick
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Thermus Thermophilus ◽  
Critical Role ◽  
Enzyme Analysis ◽  
Dna Uptake ◽  
Dna And Rna ◽  
Terminal Domains

Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus thermophilus) is required for high transformation efficiency, but its mechanism of action is unknown. In the present study, we show that TtPilF is able to bind to both DNA and RNA. The structure of TtPilF was determined by cryoelectron microscopy in the presence and absence of the ATP analogue p[NH]ppA (adenosine 5′-[β,γ-imido]triphosphate), at 10 and 12 Å (1 Å=0.1 nm) resolutions respectively. It consists of two distinct N- and C-terminal regions, separated by a short stem-like structure. Binding of p[NH]ppA induces structural changes in the C-terminal domains, which are transmitted via the stem to the N-terminal domains. Molecular models were generated for the apoenzyme and p[NH]ppA-bound states in the C-terminal regions by docking of a model based on a crystal structure from a closely related enzyme. Analysis of DNA binding by electron microscopy, using gold labelling, localized the binding site to the N-terminal domains. The results suggest a model in which DNA uptake by TtPilF is powered by ATP hydrolysis, causing conformational changes in the C-terminal domains, which are transmitted via the stem to take up DNA into the cell.

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