Conformational Changes in Myosin under ATP Hydrolysis

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

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Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

Bioinformatics and Biology Insights ◽

10.4137/bbi.s9902 ◽

2012 ◽

Vol 6 ◽

pp. BBI.S9902 ◽

Cited By ~ 3

Author(s):

Divya P. Syamaladevi ◽

Margaret S Sunitha ◽

S. Kalaimathy ◽

Chandrashekar C. Reddy ◽

Mohammed Iftekhar ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Homo Sapiens ◽

Relevant Literature ◽

Myosin Ii ◽

Coiled Coil ◽

Structural Features ◽

Model Organisms ◽

Congenital Diseases ◽

C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

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A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin

Cell ◽

10.1016/s0092-8674(00)00090-8 ◽

2000 ◽

Vol 102 (5) ◽

pp. 683-694 ◽

Cited By ~ 105

Author(s):

William M Shih ◽

Zygmunt Gryczynski ◽

Joseph R Lakowicz ◽

James A Spudich

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Molecular Motor

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Conformational Changes Generated in GroEL during ATP Hydrolysis as Seen by Time-resolved Infrared Spectroscopy

Journal of Biological Chemistry ◽

10.1074/jbc.274.9.5508 ◽

1999 ◽

Vol 274 (9) ◽

pp. 5508-5513 ◽

Cited By ~ 19

Author(s):

Frithjof von Germar ◽

Asier Galán ◽

Oscar Llorca ◽

Jose L. Carrascosa ◽

Jose M. Valpuesta ◽

...

Keyword(s):

Infrared Spectroscopy ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Time Resolved ◽

Time Resolved Infrared Spectroscopy

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Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

eLife ◽

10.7554/elife.21510 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 43

Author(s):

Marcel J Tauchert ◽

Jean-Baptiste Fourmann ◽

Reinhard Lührmann ◽

Ralf Ficner

Keyword(s):

Conformational Changes ◽

Bound States ◽

Rna Binding ◽

Atp Hydrolysis ◽

Bound State ◽

Rna Helicases ◽

Large Rearrangements ◽

Rna Complex ◽

Structural Insights ◽

Terminal Domains

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.

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ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

Biochemical Journal ◽

10.1042/bcj20190736 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3737-3750 ◽

Cited By ~ 5

Author(s):

Sabrina Lusvarghi ◽

Suresh V. Ambudkar

Keyword(s):

Conformational Changes ◽

Drug Transport ◽

Atp Hydrolysis ◽

Atp Binding ◽

Dependent Manner ◽

Deficient Mutant ◽

Concentration Dependent Manner ◽

Glycoprotein P ◽

Binding Domains ◽

P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

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The conformational changes coupling ATP hydrolysis and translocation in a bacterial DnaB helicase

Nature Communications ◽

10.1038/s41467-018-07968-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 17

Author(s):

Thomas Wiegand ◽

Riccardo Cadalbert ◽

Denis Lacabanne ◽

Joanna Timmins ◽

Laurent Terradot ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Dnab Helicase

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Structural basis for DEAH-helicase activation by G-patch proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913880117 ◽

2020 ◽

Vol 117 (13) ◽

pp. 7159-7170 ◽

Cited By ~ 2

Author(s):

Michael K. Studer ◽

Lazar Ivanović ◽

Marco E. Weber ◽

Sabrina Marti ◽

Stefanie Jonas

Keyword(s):

Conformational Changes ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Adenosine Diphosphate ◽

Structural Basis ◽

Rna Helicases ◽

Catalytic Core ◽

Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

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G-actin conformational change and polymerization induced by paraquat

Biochemistry and Cell Biology ◽

10.1139/o98-019 ◽

1998 ◽

Vol 76 (4) ◽

pp. 583-591 ◽

Cited By ~ 3

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.

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