The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

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The mechanism of kinesin inhibition by kinesin-binding protein

eLife ◽

10.7554/elife.61481 ◽

2020 ◽

Vol 9 ◽

Author(s):

Joseph Atherton ◽

Jessica JA Hummel ◽

Natacha Olieric ◽

Julia Locke ◽

Alejandro Peña ◽

...

Keyword(s):

Cell Function ◽

Binding Protein ◽

Eukaryotic Cell ◽

Motor Domain ◽

Tetratricopeptide Repeat ◽

Kinesin Motor ◽

Temporal Regulation ◽

Cryo Electron Microscopy ◽

Local Environments ◽

Binding Surface

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP–kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

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Domain topology of human Rasal

Biological Chemistry ◽

10.1515/hsz-2017-0159 ◽

2017 ◽

Vol 399 (1) ◽

pp. 63-72 ◽

Cited By ~ 2

Author(s):

Jorge Cuellar ◽

José María Valpuesta ◽

Alfred Wittinghofer ◽

Begoña Sot

Keyword(s):

Electron Microscopy ◽

Small Gtpase ◽

Full Length ◽

Gtpase Activating Protein ◽

Ras Gtpase ◽

Cryo Electron Microscopy ◽

Domain Protein ◽

Negative Staining Electron Microscopy ◽

Resolution Structure

AbstractRasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BPand Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BPand Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

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Domains in the 1α Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas

The Journal of Cell Biology ◽

10.1083/jcb.146.4.801 ◽

1999 ◽

Vol 146 (4) ◽

pp. 801-818 ◽

Cited By ~ 57

Author(s):

Steven H. Myster ◽

Julie A. Knott ◽

Katrina M. Wysocki ◽

Eileen O'Toole ◽

Mary E. Porter

Keyword(s):

Heavy Chain ◽

Motor Domain ◽

Flagellar Motility ◽

Phosphate Binding ◽

Binding Motifs ◽

Phototactic Behavior ◽

Dynein Heavy Chain ◽

Dynein Motor ◽

Mutant Phenotypes

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1α Dhc of the I1 inner dynein arm. The 1α Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH2- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1α Dhcs were recovered. The truncated 1α Dhc fragments lacked the dynein motor domain, but still assembled with the 1β Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1α motor domain in the I1 structure and further revealed the role of the 1α Dhc in flagellar motility and phototactic behavior.

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Near-atomic cryo-EM structure of yeast kinesin-5-microtubule complex reveals a distinct binding footprint

10.1101/302455 ◽

2018 ◽

Author(s):

Ottilie von Loeffelholz ◽

Alejandro Peña ◽

Douglas Robert Drummond ◽

Robert Cross ◽

Carolyn Ann Moores

Keyword(s):

Electron Microscopy ◽

Cell Division ◽

Fission Yeast ◽

Atomic Structure ◽

Binding Pocket ◽

Drug Binding ◽

Motor Domain ◽

List Type ◽

The Core ◽

Cryo Electron Microscopy

SummaryKinesin-5s are essential members of the superfamily of microtubule-dependent motors that undertake conserved roles in cell division. We investigated coevolution of the motor-microtubule interface using cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to fission yeast microtubules. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation, with a closed nucleotide binding pocket and docked neck linker that supports cover neck bundle formation. Compared to mammalian tubulin microtubules, Cut7’s footprint on S. pombe microtubule surface is subtly different because of their different architecture. However, the core motor-microtubule interaction that stimulates motor ATPase is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. The S. pombe microtubules were bound by the drug epothilone, which is visible in the taxane binding pocket. Stabilization of S. pombe microtubules is mediated by drug binding at this conserved site despite their noncanonical architecture and mechanochemistry.HighlightsS. pombe Cut7 has a distinct binding footprint on S. pombe microtubulesThe core interface driving microtubule activation of motor ATPase is conservedThe neck linker is docked in AMPPNP-bound Cut7 and the cover neck bundle is formedEpothilone binds at the taxane binding site to stabilize S. pombe microtubuleseTOC textTo investigate coevolution of the motor-microtubule interface, we used cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to microtubules polymerized from natively purified fission yeast tubulin and stabilised by the drug epothilone.

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The mechanism of selective kinesin inhibition by kinesin binding protein

10.1101/2020.07.17.208736 ◽

2020 ◽

Author(s):

Joseph Atherton ◽

Jessica J. A. Hummel ◽

Natacha Olieric ◽

Julia Locke ◽

Alejandro Peña ◽

...

Keyword(s):

Cell Function ◽

Binding Protein ◽

Eukaryotic Cell ◽

Motor Domain ◽

Kinesin Motor ◽

Temporal Regulation ◽

Cryo Electron Microscopy ◽

Local Environments ◽

Binding Surface ◽

Selective Mutation

AbstractSubcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed α-solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.

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Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

Communications Biology ◽

10.1038/s42003-019-0604-2 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 16

Author(s):

Herve Celia ◽

Istvan Botos ◽

Xiaodan Ni ◽

Tara Fox ◽

Natalia De Val ◽

...

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

High Resolution ◽

Outer Membrane ◽

Motor Function ◽

Structural Information ◽

Molecular Motor ◽

Single Copy ◽

Proton Motive Force ◽

Cryo Electron Microscopy

Abstract The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

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A new model for binding of kinesin 13 to curved microtubule protofilaments

The Journal of Cell Biology ◽

10.1083/jcb.200812052 ◽

2009 ◽

Vol 185 (1) ◽

pp. 51-57 ◽

Cited By ~ 33

Author(s):

Anke M. Mulder ◽

Alex Glavis-Bloom ◽

Carolyn A. Moores ◽

Michael Wagenbach ◽

Bridget Carragher ◽

...

Keyword(s):

Electron Microscopy ◽

Adenosine Triphosphate ◽

Molecular Interactions ◽

Binding Sites ◽

Motor Proteins ◽

Motor Domain ◽

Full Length ◽

Kinesin Motor ◽

New Model ◽

Site Binding

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.

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A near atomic structure of the active human apoptosome

eLife ◽

10.7554/elife.17755 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 39

Author(s):

Tat Cheung Cheng ◽

Chuan Hong ◽

Ildikó V Akey ◽

Shujun Yuan ◽

Christopher W Akey

Keyword(s):

Electron Microscopy ◽

Cell Death ◽

Conformational Changes ◽

Atomic Structure ◽

Catalytic Domain ◽

Nucleotide Exchange ◽

Cryo Electron Microscopy ◽

Molecular Framework ◽

Death Signals

In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.

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Structural basis for the docking of mTORC1 on the lysosomal surface

Science ◽

10.1126/science.aay0166 ◽

2019 ◽

Vol 366 (6464) ◽

pp. 468-475 ◽

Cited By ~ 37

Author(s):

Kacper B. Rogala ◽

Xin Gu ◽

Jibril F. Kedir ◽

Monther Abu-Remaileh ◽

Laura F. Bianchi ◽

...

Keyword(s):

Electron Microscopy ◽

Growth Factors ◽

Protein Kinase ◽

Nucleotide Binding ◽

Target Of Rapamycin ◽

Structural Basis ◽

Mechanistic Target Of Rapamycin ◽

Rag Gtpases ◽

Cryo Electron Microscopy ◽

Guanosine Triphosphatase

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

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