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 Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor

2016 ◽  
Vol 113 (48) ◽  
pp. E7691-E7700 ◽  
Author(s):  
Nathan D. Thomsen ◽  
Michael R. Lawson ◽  
Lea B. Witkowsky ◽  
Song Qu ◽  
James M. Berger
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Ring Closure ◽  
Scattering Data ◽  
Rna Sequences ◽  
Cofactor Binding ◽  
Closed Ring ◽  
Ring Structures ◽  
Structural Methods

Ring-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 theEscherichia coliRho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering 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 structural snapshots of substepping events in a hexameric helicase/translocase.

scholarly journals Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

2019 ◽  
Vol 116 (11) ◽  
pp. 4963-4972 ◽  
Author(s):  
Igor Dikiy ◽  
Uthama R. Edupuganti ◽  
Rinat R. Abzalimov ◽  
Peter P. Borbat ◽  
Madhur Srivastava ◽  
...  
Keyword(s):  
Blue Light ◽  
Conformational Changes ◽  
Histidine Kinase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Dark State ◽  
Light Sensing

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

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.

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.

scholarly journals Crystal structures of Bax and Bak Reveal Molecular Events Initiating Apoptosis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1490-C1490
Author(s):  
Peter Czabotar ◽  
Jason Brouwer ◽  
Dana Westphal ◽  
Geoff Thompson ◽  
Peter Colman
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Molecular Mechanisms ◽  
Structural Studies ◽  
Core Domain ◽  
Hydrophobic Groove ◽  
Outer Leaflet ◽  
Molecular Events ◽  
Surface Crystal

A key event in apoptosis is the conversion of Bax or Bak from inert monomers into cytotoxic mitochondrial membrane perforating oligomers. Certain BH3-only relatives can initiate this step through direct interactions, yet the means by which conformational changes are invoked, the nature of the conformational changes themselves, the mechanism by which they insert into membranes and the process by which they perforate these barriers has largely remained a mystery. Our recent structural studies provided the first insights into this process for Bax [1]. We found that BH3 domains activate Bax by binding to a hydrophobic groove on its surface. Crystal structures of these complexes revealed an unexpected conformational change involving dissociation of a previously unrecognized "core" domain from a "latch' domain. A further structure of the freed Bax "core" domains revealed that these form dimers that possess a surface of aromatic residues which we hypothesis engage the outer leaflet of the mitochondrial membrane and induce curvature. We have now extended our studies to include structures of Bax bound to alternative BH3-only proteins providing new insights into key interactions occurring at this interface. Additionally, we have solved structures of activated Bak and of the freed Bak "core" domain dimers. These results further our understanding of the molecular mechanisms by which these highly dynamic proteins engage the mitochondrial membrane and thus control the life/death switch in cells.

scholarly journals An insightful myosin structure reveals how actin triggers force production

2014 ◽  
Vol 70 (a1) ◽  
pp. C1056-C1056
Author(s):  
Paola Llinas ◽  
Tatiana Isabet ◽  
Lin Song ◽  
Allan Zhong ◽  
Serena Sirigu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Motors ◽  
Structural State ◽  
Atp Hydrolysis ◽  
Mechanical Energy ◽  
Genetic Disorders ◽  
Force Production ◽  
Actin Binding ◽  
Mechanical Coupling ◽  
Hydrolysis Products

Directed force production is essential for life. Allostery is at the heart of the mechanism that cellular nanomotors use to walk, pull or anchor. Such molecular motors are essential for a cell to migrate, to divide and organise the intra-cellular traffic between its compartments. The actin-based motors, myosins, are critical for many of these movements, for muscle contraction, cytokinesis and sophisticated cellular functions such as hearing. Deficit in these motors can lead to a number of human genetic disorders. Force is produced by these motors by the conversion of chemical energy derived from ATP hydrolysis into mechanical energy via the interaction with their track, the actin filament. Biophysical approaches have provided insights into the chemo-mechanical coupling in the actomyosin system. They show how three allosteric sites communicate via relatively small conformational changes in the motor domain that are coupled and amplified by a lever-arm mechanism that produce a working stroke of several nanometers. While ATP binding and hydrolysis are essential for detachment of the motor from its track and its trapping in the pre-stroke conformation, step-wise rebinding to the track triggers controlled release of hydrolysis products upon the working stroke. A reverse motor, myosin VI has been particularly intriguing and informative regarding the force production mechanism. An unpublished structural state not only reveal how trapping of the hydrolysis products stabilize the primed pre-stroke conformation, it also provides insights for the rearrangements triggered by actin to promote Pi release. This new structural state has all the expected features of the Pi release state populated upon motor re-binding to its track. This allows visualization for the first time of the structural rearrangements triggered by actin binding that are coupled to force generation and product release at the beginning of the powerstroke.

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 Past, present and future experiments on muscle

2000 ◽  
Vol 355 (1396) ◽  
pp. 539-543 ◽  
Author(s):  
H. E. Huxley
Keyword(s):  
Molecular Motors ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Large Change ◽  
Technical Difficulty ◽  
Definitive Evidence ◽  
Technical Developments ◽  
Mechanical Information ◽  
Cross Bridges

Since the basic outline of the sliding filament mechanism became apparent some 45 years ago, the principal challenge, an experimental one, has been to produce definitive evidence about the detailed molecular mechanisms by which myosin cross–bridges produce force and movement in a muscle. More recently, similar questions could be posed about other molecular motors, in non–muscle cells. This problem proved unexpectedly difficult to solve, in part because of the technical difficulty of obtaining the structural and mechanical information required about rapid events within macromolecules, especially in a working system, and this triggered many remarkable technical developments. There is now very strong evidence for a large change in shape of the myosin heads during ATP hydrolysis, consistent with a leverarm mechanism. Whether this does indeed provide the driving force for contraction and movement— and, if so, exactly how—and whether some other processes could also play a significant role, is discussed in the light of the experimental and theoretical findings presented at this meeting, and other recent and long–term evidence.

scholarly journals Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

2020 ◽  
Author(s):  
Irene Maffucci ◽  
Damien Laage ◽  
Guillaume Stirnemann ◽  
Fabio Sterpone
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Conformational Sampling ◽  
Detailed Understanding ◽  
Wide Range ◽  
The Difference ◽  
Dynamics Simulations

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

scholarly journals Defects in the synthesis of human haemoglobin

1978 ◽  
Author(s):  
Ρεγγίνα Σταθοπούλου
Keyword(s):  
Structural Changes ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Red Cells ◽  
Synthesis Rate ◽  
Structural Studies ◽  
Human Haemoglobin ◽  
Abnormal Haemoglobin ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

The work described is concerned with the biosynthesis of haemoglobin in human reticulicytes and bone marrow cells with particular reference to abnormal haemoglobins. The work in abnormal haemoglobins was supported by structural studies and involved a - and ß - chain variants, some of the latter being unstable. In some of the individuals with α or ß - chain variants, as well as in a number of subjects with β- thalassaemia, the estimation of the free α - chian pool was carried out. Using the same methodology, an attempt was made to detect chain fragments in the latter group of subjects. In the α - chian variants, attention was paid to the different proportions of the same α - chian abnormal haemoglobin in different individuals. Notably so in the case of haemoglobin G Philadelphia. The results supported the interpretation that here are four α - chian genes and that the α - G Philadelphia gene may occur together with threee, two or one normal α - chian genes respectively, the number of normal α - chian genes being potentially decreased by α - thalassaemia. With ß - chain variants a number of low proportion abnormal haemoglobins in the blood was studied. The globin chain synthesis pattern in the reticulocytes was compared with the haemoglobin composition in the mature red cells and the clinical condition of the subjects. Structural changes in haemoglobin were related to the rate of its synthesis. In some instances, both the synthesised amount of the variant was low as well as its proportion in the mature red cells. In others, notably haemoglobin E and Haemoglobin newcastle, the synthesis rate of the variant was normal but its proportion in the circulating red cells was low. Possible molecular mechanisms are either instability of the variant ß - chain of defective association into functioning polymer.

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