scholarly journals Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

2021 ◽  
Author(s):  
Benjamin J LaFrance ◽  
Johanna Roostalu ◽  
Gil Henkin ◽  
Basil J Greber ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Conformational Changes ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
Structural Transitions ◽  
Gtp Hydrolysis ◽  
Tirf Microscopy ◽  
Mechanistic Insight ◽  
Catalytically Active

Microtubules (MTs) are polymers of alphabeta-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability.

scholarly journals Abstract P-33: Structural Basis for the Growing Microtubule End Recognition by End Binding Proteins

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S26-S26
Author(s):  
Alena Korshunova
Keyword(s):  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Molecular Mechanism ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
3D Structure ◽  
Structural Basis ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis

Background: Eukaryotic end binding proteins (EBs) can follow the growing microtubule end. EBs play a crucial role in microtubule dynamic instability and promote simultaneously growth rate and catastrophe frequency. It makes EB-like proteins perspective drag targets for a wide number of diseases. But the molecular mechanism of tip tracking by EB-like proteins remains unknown. Studies of mutants have revealed that the conservative amino acid Q102 (numbering relative to the human EB1 protein) plays a key role in the recognition of the growing microtubule end. However, the 3D structure studies revealed that this amino acid has no bonds with tubulin. In this work, we performed structural and phylogenetic analysis of EBs proteins to identify a possible molecular mechanism behind the plus end tracking. Methods: UCSF Chimera10 was used for structural analysis. Phylogenetic analysis was performed with MEGA X software. 3D structures of EBs and microtubules with different states of GTP hydrolysis were used (pdb 3JAK, 3JAS, 3JAT, 3JAW, 3JAL, 3JAR, 6DPU, 6DPV, 6DPW). Results: We have shown that two conservative amino acids (K100, E106) should play an important role in the recognition of the microtubule plus end in addition to Q102. It was concluded that these amino acids together form the plus-end «navigation site» of EBs. Analysis of possible interaction of the «navigation site» amino acids with microtubules in different conformational states suggested that the main mechanism of growing microtubule end recognition is not due to an affinity increase for a certain state of tubulin in microtubules at their end, but it due to a significant affinity decrease in other parts of the microtubule as a result of steric clashes. Conclusion: Thus, the results of the analysis suggested the possible molecular mechanism that provides the tip tracking by EB-like proteins and allowed us to identify the key amino acids of this mechanism.

scholarly journals Separating the effects of nucleotide and EB binding on microtubule structure

2018 ◽  
Vol 115 (27) ◽  
pp. E6191-E6200 ◽  
Author(s):  
Rui Zhang ◽  
Benjamin LaFrance ◽  
Eva Nogales
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
Stable State ◽  
Structural Data ◽  
Atomic Resolution ◽  
Mechanical Forces ◽  
Gtp Hydrolysis ◽  
Associated Proteins ◽  
Microtubule Structure

Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and are highly regulated by a number of MT-associated proteins (MAPs). How MAPs affect MTs is still not fully understood, partly due to a lack of high-resolution structural data on undecorated MTs, which need to serve as a baseline for further comparisons. Here we report three structures of MTs in different nucleotide states (GMPCPP, GDP, and GTPγS) at near-atomic resolution and in the absence of any binding proteins. These structures allowed us to differentiate the effects of nucleotide state versus MAP binding on MT structure. Kinesin binding has a small effect on the extended, GMPCPP-bound lattice, but hardly affects the compacted GDP-MT lattice, while binding of end-binding (EB) proteins can induce lattice compaction (together with lattice twist) in MTs that were initially in an extended and more stable state. We propose a MT lattice-centric model in which the MT lattice serves as a platform that integrates internal tubulin signals, such as nucleotide state, with outside signals, such as binding of MAPs or mechanical forces, resulting in global lattice rearrangements that in turn affect the affinity of other MT partners and result in the exquisite regulation of MT dynamics.

scholarly journals Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

2022 ◽  
Vol 119 (2) ◽  
pp. e2114994119
Author(s):  
Benjamin J. LaFrance ◽  
Johanna Roostalu ◽  
Gil Henkin ◽  
Basil J. Greber ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Total Internal Reflection ◽  
Dynamic Instability ◽  
Wild Type ◽  
Gtp Hydrolysis ◽  
Cryo Electron Microscopy ◽  
Catalytically Active ◽  
The Stability ◽  
Insight Into

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

scholarly journals High-Resolution Microtubule Structures Reveal the Structural Transitions in αβ-Tubulin upon GTP Hydrolysis

Cell ◽  
2014 ◽  
Vol 157 (5) ◽  
pp. 1117-1129 ◽  
Author(s):  
Gregory M. Alushin ◽  
Gabriel C. Lander ◽  
Elizabeth H. Kellogg ◽  
Rui Zhang ◽  
David Baker ◽  
...  
Keyword(s):  
High Resolution ◽  
Structural Transitions ◽  
Gtp Hydrolysis

scholarly journals Abstract P-36: Structural Analysis of Conformational Changes of Bacterial and Eukaryotic Tubulins

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S27-S28
Author(s):  
Liubov Makarova ◽  
Alena Korshunova
Keyword(s):  
Structural Analysis ◽  
Conformational Changes ◽  
Structural Changes ◽  
Dynamic Instability ◽  
Amino Acid Sequences ◽  
Anchor Point ◽  
Final State ◽  
Gtp Hydrolysis ◽  
Alpha Tubulin ◽  
Β Tubulin

Background: Eukaryotic α- and β-tubulin proteins stand out among tubulin-like proteins by their ability to form hollow dynamically unstable microtubules (MT) with 13 protofilaments. Microtubules are part of the cell cytoskeleton and play a key role in chromosome division in mitosis. A considerable amount of anticancer drugs works on microtubules level breaking its dynamic. But the mechanism of dynamic instability and works of these drugs remains unknown. Bacteria of the genus Prostecobacter have unique bacterial tubulins (BtubA/B) capable to form hollow dynamically unstable 5 protofilament MTs (miniMT). Instead of great differences, both tubulins have many common features. Eukaryotic tubulin was known to have structural changes through GTP hydrolysis (compactization for approximately 2 Å and a twist for 0,1˚). «Anchor point» structure in alpha-tubulin was noticed to be a fixed point in this movement. Methods: We performed comparative structural analysis of BtubA/B and α- and β-tubulin proteins using USCF Chimera10 and MEGA X software. This data was obtained due to a comparison of 3 structures of microtubules with different nucleotides [pdb6DPU, 6DPV, 6DPW] and two structures for bacterial tubulins (miniMT [pdb5o09] and BtubA/B-dimer [pdb2BTQ]). Results: We noticed that bacterial tubulins form shorter protofilaments in miniMT than eukaryotic ones. It can be explained as compaction in two sites instead of one site in eukaryotic MT. Also, the most motionless point of min MT turned out the same "anchor point." Phylogenetic analysis showed that this structure is very conservative in these orthologs. Moreover, the final state of both tubulins (GDP) repeats each other. Conclusion: Our results suggest that bacterial tubulin can have movements through GTP hydrolysis similar to eukaryotic one. And it means that despite different amino acid sequences, bacterial and eukaryotic tubulins have similar keys structures for dynamic instability.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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

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.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

2019 ◽  
Vol 19 (22) ◽  
pp. 1952-1961 ◽  
Author(s):  
J.C. Sobrinho ◽  
A.F. Francisco ◽  
R. Simões-Silva ◽  
A.M. Kayano ◽  
J.J. Alfonso Ruiz Diaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Synthetic Peptides ◽  
Cell Damage ◽  
Neutralization Assay ◽  
Phospholipase Activity ◽  
Phospholipases A2 ◽  
Catalytically Active ◽  
Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

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

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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