High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.

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High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

10.1101/2021.09.07.459262 ◽

2021 ◽

Author(s):

Sabrina Pospich ◽

H. Lee Sweeney ◽

Anne Houdusse ◽

Stefan Raunser

Keyword(s):

High Resolution ◽

Bound States ◽

Structural Model ◽

Molecular Motor ◽

Structural Data ◽

Underlying Mechanism ◽

Actin Binding ◽

Structural Transitions ◽

Myosin V ◽

Motor Cycle

AbstractThe molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

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Converter domain mutations in myosin alter structural kinetics and motor function

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006128 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1554-1567 ◽

Cited By ~ 5

Author(s):

Laura K. Gunther ◽

John A. Rohde ◽

Wanjian Tang ◽

Shane D. Walton ◽

William C. Unrath ◽

...

Keyword(s):

Motor Function ◽

Rate Constants ◽

Molecular Motors ◽

Atp Hydrolysis ◽

Actin Binding ◽

Power Stroke ◽

Structural Transitions ◽

Myosin V ◽

Time Resolved

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility–sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power–stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power–stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power–stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

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Studies of Conformational Changes of Tubulin Induced by Interaction with Kinesin Using Atomistic Molecular Dynamics Simulations

International Journal of Molecular Sciences ◽

10.3390/ijms22136709 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6709

Author(s):

Xiao-Xuan Shi ◽

Peng-Ye Wang ◽

Hong Chen ◽

Ping Xie

Keyword(s):

Molecular Dynamics ◽

High Resolution ◽

Binding Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Weak Interactions ◽

Molecular Motor ◽

Structural Data ◽

Calculated Binding Energy ◽

Dynamics Simulations

The transition between strong and weak interactions of the kinesin head with the microtubule, which is regulated by the change of the nucleotide state of the head, is indispensable for the processive motion of the kinesin molecular motor on the microtubule. Here, using all-atom molecular dynamics simulations, the interactions between the kinesin head and tubulin are studied on the basis of the available high-resolution structural data. We found that the strong interaction can induce rapid large conformational changes of the tubulin, whereas the weak interaction cannot. Furthermore, we found that the large conformational changes of the tubulin have a significant effect on the interaction of the tubulin with the head in the weak-microtubule-binding ADP state. The calculated binding energy of the ADP-bound head to the tubulin with the large conformational changes is only about half that of the tubulin without the conformational changes.

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Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Nature Communications ◽

10.1038/s41467-021-26848-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hengjun Cui ◽

Andreas U. Müller ◽

Marc Leibundgut ◽

Jiawen Tian ◽

Nenad Ban ◽

...

Keyword(s):

High Resolution ◽

Sequence Homology ◽

Active Site ◽

Bound States ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Reaction Pathway ◽

Post Translational Modification ◽

Functional States

AbstractPupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

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Structural insights into filament recognition by cellular actin markers

10.1101/846337 ◽

2019 ◽

Cited By ~ 3

Author(s):

Archana Kumari ◽

Shubham Kesarwani ◽

Manjunath G Javoor ◽

Kutti R. Vinothkumar ◽

Minhajuddin Sirajuddin

Keyword(s):

High Resolution ◽

Binding Sites ◽

Binding Proteins ◽

Structural Model ◽

Structural Data ◽

Actin Binding ◽

Filamentous Actin ◽

Electron Cryomicroscopy ◽

Structural Insights ◽

Structural Perspective

AbstractCellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

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Myosin V executes steps of variable length via structurally constrained diffusion

eLife ◽

10.7554/elife.51569 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

David Hathcock ◽

Riina Tehver ◽

Michael Hinczewski ◽

D Thirumalai

Keyword(s):

Binding Sites ◽

Actin Filaments ◽

Recent Experiment ◽

Molecular Motor ◽

Biological Significance ◽

Actin Binding ◽

Size Distributions ◽

Myosin V ◽

Step Size ◽

Load Dependence

The molecular motor myosin V transports cargo by stepping on actin filaments, executing a random diffusive search for actin binding sites at each step. A recent experiment suggests that the joint between the myosin lever arms may not rotate freely, as assumed in earlier studies, but instead has a preferred angle giving rise to structurally constrained diffusion. We address this controversy through comprehensive analytical and numerical modeling of myosin V diffusion and stepping. When the joint is constrained, our model reproduces the experimentally observed diffusion, allowing us to estimate bounds on the constraint energy. We also test the consistency between the constrained diffusion model and previous measurements of step size distributions and the load dependence of various observable quantities. The theory lets us address the biological significance of the constrained joint and provides testable predictions of new myosin behaviors, including the stomp distribution and the run length under off-axis force.

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Systematic Study of the Functions for the Residues around the Nucleotide Pocket in Simian Virus 40 AAA+ Hexameric Helicase

Journal of Virology ◽

10.1128/jvi.00387-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 6017-6023 ◽

Cited By ~ 23

Author(s):

William B. Greenleaf ◽

Jingping Shen ◽

Dahai Gai ◽

Xiaojiang S. Chen

Keyword(s):

Atp Hydrolysis ◽

Mutational Analysis ◽

Molecular Motor ◽

Simian Virus 40 ◽

Structural Data ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

New Type ◽

Arginine Finger

ABSTRACT The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the γ-phosphate of ATP or the apical water molecule. A potentially noncanonical “arginine finger” residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.

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High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122964 ◽

1992 ◽

Vol 50 (1) ◽

pp. 512-513

Author(s):

J. Jakana ◽

M.F. Schmid ◽

P. Matsudaira ◽

W. Chiu

Keyword(s):

High Resolution ◽

Binding Proteins ◽

Actin Binding ◽

Eukaryotic Cells ◽

Dimensional Structure ◽

Structural Basis ◽

Actin Bundle ◽

Actin Bundles ◽

3 Dimensional ◽

Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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Tubulin structure at intermediate resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140634 ◽

1995 ◽

Vol 53 ◽

pp. 852-853

Author(s):

K. H. Downing ◽

S. G. Wolf ◽

E. Nogales

Keyword(s):

High Resolution ◽

Structural Data ◽

Therapeutic Drug ◽

Zinc Ions ◽

Two Dimensional ◽

X Ray Diffraction ◽

X Ray ◽

Negative Stain ◽

Functional Mechanisms ◽

Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

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HRTEM study of valleriite, a hydroxide-bearing copper sulfide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100175442 ◽

1990 ◽

Vol 48 (4) ◽

pp. 462-463

Author(s):

S. Wang ◽

P. R. Buseck

Keyword(s):

Electron Microscope ◽

High Resolution ◽

Copper Sulfide ◽

Carbonaceous Chondrite ◽

Structural Data ◽

Basic Structure ◽

Long Period ◽

Selected Area Electron Diffraction ◽

Transmission Electron ◽

Wavy Structure

Valleriite is an unusual mineral, consisting of intergrowths of sulfide layers (corresponding in structure to the mineral smythite - Fe9S11) and hydroxide layers (corresponding to brucite - Mg(OH2)). It has a composition of approximately 1.526[Mg.68Al.32(OH)2].[Fe1.07Cu.93S2] and consists of two interpenetrating lattices, each of which retains its individual structural and diffraction characteristics parallel to the layering. The valleriite structure is related to that of tochilinite, an unusual iron-rich mineral that is of considerable interest for the origin of certain carbonaceous chondrite meteorites and to those of franckeite and cylindrite, two minerals that are of interest because of their unique morphological and crystallographic properties, e.g., the distinctive curved form of cylindrite and the perfect mica-like cleavage with unusual striations and the long-period wavy structure of franckeite.Our selected-area electron diffraction (SAED) patterns and high-resolution transmission electron microscope (HRTEM) images of valleriite provide new structural data. A basic structure and a new superstructure have been observed.

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