scholarly journals Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble

2020 ◽  
Vol 21 (8) ◽  
pp. 2843
Author(s):  
Kenta Ishibashi ◽  
Hitoshi Sakakibara ◽  
Kazuhiro Oiwa
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Current Knowledge ◽  
Expression System ◽  
Molecular Architecture ◽  
Electron Microscopic ◽  
Axonemal Dynein ◽  
Cilia And Flagella ◽  
Amount Of Knowledge

In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.

scholarly journals How signals of calcium ions initiate the beats of cilia and flagella

2019 ◽  
Author(s):  
M. V. Satarić ◽  
T. Nemeš ◽  
D. Sekulić ◽  
J. A. Tuszynski
Keyword(s):  
Atp Hydrolysis ◽  
Cellular Motility ◽  
Cell Organelles ◽  
Ciliary Movement ◽  
Ciliary Function ◽  
Dynein Motor ◽  
Combined Action ◽  
Axonemal Dynein ◽  
Cilia And Flagella

ABSTRACTCilia and flagella are cell organelles serving basic roles in cellular motility. Ciliary movement is performed by a sweeping-like repeated bending motion, which gives rise to a self-propagating “ciliary beat”. The hallmark structure in cilia is the axoneme, a stable architecture of microtubule doublets. The motion of axoneme is powered by the axonemal dynein motor family powered by ATP hydrolysis. It is still unclear how the organized beat of cilium and flagella emerges from the combined action of hundreds of dynein molecules. It has been hypothesized that such coordination is mediated by mechanical stress due to transverse, radial or sliding deformations. The beating asymmetry is crucial for airway ciliary function and it requires tubulin glutamination a unique posttranslational modification of C-termini of constituent microtubules that is highly abundant in cilia and flagella. The exact role of tubulin glutamination in ciliary or flagellar function is still unclear. Here we examine the role of calcium (Ca2+) ions based on the experimental evidences that the flagellar asymmetry can be increased due to the entry of extracellular Ca2+through, for example, nimodipine-sensitive pathway located in the flagella. We propose a new scenario based on the polyelectrolyte properties of cellular microtubules (MTs) such that dynamic influx of Ca2+ions provides the initiation and synchronization of dynein sliding along microtubules. We also point out the possible interplay between tubulin polyglutaminated C-termini and localized pulses of Ca2+ions along microtubules.

scholarly journals Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis

2018 ◽  
Vol 4 (10) ◽  
pp. eaat8797 ◽  
Author(s):  
Nagaraju Chada ◽  
Kanokporn Chattrakun ◽  
Brendan P. Marsh ◽  
Chunfeng Mao ◽  
Priya Bariya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Real Space ◽  
Biochemical Activity ◽  
Force Microscopy ◽  
Atomic Force ◽  
Reversible Transitions ◽  
Space Measurement

SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.

scholarly journals HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

2019 ◽  
Author(s):  
Zainab Ahdash ◽  
Euan Pyle ◽  
William J. Allen ◽  
Robin A. Corey ◽  
Ian Collinson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Conformational Dynamics ◽  
Brownian Ratchet ◽  
Exchange Mass Spectrometry ◽  
Dependent Transport ◽  
Hdx Ms

AbstractThe bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Samuel E Lacey ◽  
Shaoda He ◽  
Sjors HW Scheres ◽  
Andrew P Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

scholarly journals CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gerard W. Dougherty ◽  
Katsutoshi Mizuno ◽  
Tabea Nöthe-Menchen ◽  
Yayoi Ikawa ◽  
Karsten Boldt ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Adenine Nucleotide ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Situs Inversus Totalis ◽  
Proteomic Profiling ◽  
Binding Interface ◽  
Axonemal Dynein ◽  
Cilia And Flagella

Abstract Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

2019 ◽  
Author(s):  
Samuel E. Lacey ◽  
Shaoda He ◽  
Sjors H. W. Scheres ◽  
Andrew P. Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

AbstractDyneins are motor proteins responsible for transport in the cytoplasm and the beating of the axoneme in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a long coiled-coil stalk. Here we address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using the stand-alone Relion package. We show the majority of the MTBD is remarkably rigid upon binding, with the transition to the high affinity state controlled by the movement of a single helix at the MTBD interface. In addition DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that reaches over and contacts the adjacent protofilament. Unexpectedly we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer

2005 ◽  
Vol 33 (4) ◽  
pp. 878-882 ◽  
Author(s):  
M. Börsch ◽  
P. Gräber
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Atp Synthesis ◽  
Fluorescence Resonance ◽  
Atp Synthases

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.

scholarly journals Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ling Wang ◽  
Zachary Lee Johnson ◽  
Michael R Wasserman ◽  
Jesper Levring ◽  
Jue Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Multidrug Resistance Protein 1 ◽  
Rate Limiting Step ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Resistance Protein ◽  
Rate Limiting

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling

2011 ◽  
Vol 39 (2) ◽  
pp. 611-616 ◽  
Author(s):  
Dagmar Klostermeier
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Machines ◽  
Dna Supercoiling ◽  
Single Molecule Fret ◽  
Gyrase A ◽  
Rna Unwinding

Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.

Molecular architecture of bacterial flagellum

1997 ◽  
Vol 30 (1) ◽  
pp. 1-65 ◽  
Author(s):  
KEIICHI NAMBA ◽  
FERENC VONDERVISZT
Keyword(s):  
Conformational Changes ◽  
Structural Design ◽  
Structural Motif ◽  
Molecular Architecture ◽  
Electron Microscopic ◽  
Reconstruction Procedure ◽  
X Ray ◽  
Chain Folding ◽  
Bacterial Flagellum

1. INTRODUCTION 22. OVERALL STRUCTURE AND SUBSTRUCTURES 52.1 Overall structure and components 52.2 Bidirectional rotary motor 52.3 Drive shaft 82.4 Bushing 82.5 Universal joint 92.6 Helical propeller 92.7 Axial junction 102.8 Capping structure 113. ASSEMBLY PROCESS OF THE FLAGELLUM 113.1 Step by step assembly 113.2 Flagellum-specific export apparatus and the channel 124. UNIQUE CHARACTERISTICS OF THE FLAGELLAR MOTOR DYNAMICS 135. STRUCTURAL DESIGN OF FLAGELLIN FOR ASSEMBLY REGULATION AND POLYMORPHISM 145.1 Domain organization and terminal disorder of flagellin 155.2 The role of terminal disorder in filament formation and polymorphism 175.3 Common structural motif for regulation of self-assembly 216. STRUCTURAL DESIGN OF FLAGELLAR FILAMENTS FOR POLYMORPHISM 226.1 Polymorphic mechanism 236.2 Structures of the filaments deduced by electron microscopy 256.2.1 Overview of the electron microscopic studies 256.2.2 Helical image reconstruction procedure 276.2.3 Structural details of the filament 286.3 X-ray fibre diffraction studies 326.3.1 Overview of the X-ray studies 326.3.2 Orientation of liquid crystalline sols and diffraction patterns 336.3.3 Equatorial analysis 356.3.4 A preliminary map refined at 11 Åresolution 376.4 Overall chain folding of the subunit in the filament 386.4.1 Mapping out the terminal and central regions 386.4.2 The chain folding and role of each domain 426.5 Polymorphic nature of flagellar filament 436.5.1 Comparison of the L- and R-type 436.5.2 New helical symmetry ‘Lt-type’ 466.5.3 Direct comparison of the Lt-type lattice to the other two 486.5.4 Plausible conformational changes involved in polymorphism 517. PERSPECTIVE 558. ACKNOWLEDGEMENTS 559. REFERENCES 55

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