scholarly journals Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition

2018 ◽  
Vol 115 (51) ◽  
pp. E11933-E11942 ◽  
Author(s):  
Jinqi Ren ◽  
Shuang Wang ◽  
Han Chen ◽  
Wenjuan Wang ◽  
Lin Huo ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Underlying Mechanism ◽  
Motor Domain ◽  
Dimer Formation ◽  
Interaction Interface ◽  
Adp Release ◽  
Interdomain Interaction ◽  
Neck Domain ◽  
Neck Coil

In kinesin-3, the coiled-coil 1 (CC1) can sequester the preceding neck coil (NC) for autoinhibition, but the underlying mechanism is poorly understood. Here, we determined the structures of the uninhibited motor domain (MD)-NC dimer and inhibited MD-NC-CC1 monomer of kinesin-3 KIF13B. In the MD-NC-CC1 monomer, CC1 is broken into two short helices that unexpectedly interact with both the NC and the MD. Compared with the MD-NC dimer, the CC1-mediated integration of NC and MD not only blocks the NC dimer formation, but also prevents the neck linker (NL) undocking and the ADP release from the MD. Mutations of the essential residues in the interdomain interaction interface in the MD-NC-CC1 monomer restored the MD activity. Thus, CC1 fastens the neck domain and MD and inhibits both NC and NL. This CC1-mediated lockdown of the entire neck domain may represent a paradigm for kinesin autoinhibition that could be applicable to other kinesin-3 motors.

scholarly journals Prominin-1 Modulates Rho/ROCK-Mediated Membrane Morphology and Calcium-Dependent Intracellular Chloride Flux

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Akiko Hori ◽  
Kenji Nishide ◽  
Yuki Yasukuni ◽  
Kei Haga ◽  
Wataru Kakuta ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Coiled Coil ◽  
Chloride Ion ◽  
Underlying Mechanism ◽  
Mouse Embryonic Fibroblast ◽  
Small Gtpase ◽  
Calcium Dependent ◽  
Membrane Morphology ◽  
Fibre Formation ◽  
Amino Acid Stretch

Abstract Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.

Retention of Coiled-Coil Dimer Formation in the Absence of Ion Pairing at Positions Flanking the Hydrophobic Core

Biochemistry ◽  
2019 ◽  
Vol 58 (48) ◽  
pp. 4821-4826 ◽  
Author(s):  
Naomi A. Biok ◽  
Alexander D. Passow ◽  
Chenxuan Wang ◽  
Craig A. Bingman ◽  
Nicholas L. Abbott ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Ion Pairing ◽  
Hydrophobic Core ◽  
Dimer Formation

scholarly journals The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

2005 ◽  
Vol 16 (8) ◽  
pp. 3529-3537 ◽  
Author(s):  
Friederike Bathe ◽  
Katrin Hahlen ◽  
Renate Dombi ◽  
Lucia Driller ◽  
Manfred Schliwa ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Coiled Coil ◽  
Proximal Part ◽  
Tail Domain ◽  
Yeast Two Hybrid ◽  
Complex Interplay ◽  
Hybrid Data ◽  
Neck Domain ◽  
Two Hybrid ◽  
Complex Manner

Kinesin-1 dimerizes via the coiled-coil neck domain. In contrast to animal kinesins, neck dimerization of the fungal kinesin-1 NcKin requires additional residues from the hinge. Using chimeric constructs containing or lacking fungal-specific elements, the proximal part of the hinge was shown to stabilize the neck coiled-coil conformation in a complex manner. The conserved fungal kinesin hinge residue W384 caused neck coiled-coil formation in a chimeric NcKin construct, including parts of the human kinesin-1 stalk. The stabilizing effect was retained in a NcKinW384F mutant, suggesting important π -stacking interactions. Without the stalk, W384 was not sufficient to induce coiled-coil formation, indicating that W384 is part of a cluster of several residues required for neck coiled-coil folding. A W384-less chimera of NcKin and human kinesin possessed a non–coiled-coil neck conformation and showed inhibited activity that could be reactivated when artificial interstrand disulfide bonds were used to stabilize the neck coiled-coil conformation. On the basis of yeast two-hybrid data, we propose that the proximal hinge can bind kinesin's cargo-free tail domain and causes inactivation of kinesin by disrupting the neck coiled-coil conformation.

scholarly journals The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

2020 ◽  
Author(s):  
Fangfang Jiang ◽  
Yasuharu Takagi ◽  
Arik Shams ◽  
Sarah M. Heissler ◽  
Thomas B. Friedman ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Motor Domain ◽  
Actin Binding ◽  
Duty Ratio ◽  
Strain Sensing ◽  
Cochlear Hair Cells ◽  
Adp Release ◽  
Chemical Cycle ◽  
Thermodynamic Coupling ◽  
Rate Limit

AbstractCochlear hair cells possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3. To understand the molecular mechanism of how MYO15A delivers proteins within stereocilia, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B co-expression had similar ATPase activities (kcat = ~ 6 s−1 at 20°C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP and actin binding, hydrolysis and phosphate release. Actin-attached ADP release was the slowest measured transition (~ 12 s−1 at 20°C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (~ 0.5) and weak thermodynamic coupling between ADP and actin binding. This is consistent with MYO15 being adapted for strain sensing as a monomer, or processive motility if oligomerized into ensembles. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and ultimately disrupt stereocilia trafficking necessary for normal hearing.

scholarly journals Coiled-Coil Domain Containing 80 Suppresses Nonylphenol-Induced Colorectal Cancer Cell Proliferation by Inhibiting the Activation of ERK1/2

2021 ◽  
Vol 9 ◽  
Author(s):  
Jing Wang ◽  
Yuan-wei Zhang ◽  
Nian-jie Zhang ◽  
Shuo Yin ◽  
Du-ji Ruan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Differentially Expressed Genes ◽  
Endocrine Disrupting Chemicals ◽  
Coiled Coil ◽  
Underlying Mechanism ◽  
Differentially Expressed ◽  
Coiled Coil Domain ◽  
Upregulated Genes

Recently, the effect of endocrine-disrupting chemicals on the cancer procession has been a concern. Nonylphenol (NP) is a common environmental estrogen that has been shown to enhance the proliferation of colorectal cancer (CRC) cells in our previous studies; however, the underlying mechanism remains unclear. In this study, we confirmed the increased concentration of NP in the serum of patients with CRC. RNA sequencing was used to explore the differentially expressed genes after NP exposure. We found 16 upregulated genes and 12 downregulated genes in COLO205 cells after NP treatment. Among these differentially expressed genes, we found that coiled-coil domain containing 80 (CCDC80) was downregulated by NP treatment and was associated with CRC progression. Further experiments revealed that the overexpression of CCDC80 significantly suppressed NP-induced cell proliferation and recovered the reduced cell apoptosis. Meanwhile, the overexpression of CCDC80 significantly inhibited the activation of ERK1/2 induced by NP treatment. ERK1/2 inhibitor (PD98059) treatment also suppressed NP-induced CRC cell growth, but the overexpression of CCDC80 did not enhance the effect of ERK1/2 inhibitor. Taken together, NP treatment significantly inhibited the expression of CCDC80, and the overexpression of CCDC80 suppressed NP-induced CRC cell growth by inhibiting the activation of ERK1/2. These results suggest that NP could induce CRC cell growth by influencing the expression of multiple genes. CCDC80 and ERK1/2 inhibitors may be suitable therapeutic targets in NP-related CRC progression.

scholarly journals Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport.

1994 ◽  
Vol 127 (4) ◽  
pp. 1041-1048 ◽  
Author(s):  
P A Pesavento ◽  
R J Stewart ◽  
L S Goldstein
Keyword(s):  
Nervous System ◽  
Axonal Transport ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Coiled Coil ◽  
Biochemical Properties ◽  
Motor Domain ◽  
Tail Region ◽  
Anterograde Axonal Transport ◽  
Kinesin Heavy Chain

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.

scholarly journals Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia

2007 ◽  
Vol 402 (2) ◽  
pp. 349-357 ◽  
Author(s):  
Takashi Murayama ◽  
Toshiharu Oba ◽  
Hiroshi Hara ◽  
Kikuo Wakebe ◽  
Noriaki Ikemoto ◽  
...  
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Adenine Nucleotide ◽  
Underlying Mechanism ◽  
Mammalian Skeletal Muscle ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Fk506 Binding Protein ◽  
Interdomain Interaction

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle (‘suppression’ of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222–C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615→Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442–Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.

scholarly journals Coiled-coil registry shifts in the F684I mutant of Bicaudal result in cargo-independent activation of dynein motility

2019 ◽  
Author(s):  
Heying Cui ◽  
Kathleen M. Trybus ◽  
M. Yusuf Ali ◽  
Puja Goyal ◽  
Kaiqi Zhang ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Underlying Mechanism ◽  
Structural Flexibility ◽  
Human Homolog ◽  
Wild Type ◽  
Transport Pathways ◽  
X Ray ◽  
In The Wild ◽  
Selection For ◽  
Dependent Transport

ABSTRACTThe dynein adaptor Drosophila Bicaudal D (BicD) is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full-length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X-ray structure of the C-terminal domain of the BicD/F684I mutant reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled-coil registry shift upon cargo binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2–dependent transport pathways.

scholarly journals Dexamethasone inhibits U937 cell adhesion via the down-regulation of ROCK1 activity.

2012 ◽  
Vol 59 (4) ◽  
Author(s):  
Dong Liu ◽  
Xing-Yun Chen ◽  
Ren-Ping Xiong ◽  
Ya-Lei Ning ◽  
Ping Li ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
U937 Cell ◽  
Umbilical Vein ◽  
Coiled Coil ◽  
Underlying Mechanism ◽  
U937 Cells ◽  
Monocyte Adhesion ◽  
Myristate Acetate ◽  
Ru 486 ◽  
Umbilical Vein Endothelial Cells

To explore the effect of dexamethasone (DEX) on monocyte adhesion function and its underlying mechanism. The effects of DEX and fasudil on adhesion of cultured U937 monocytes to human umbilical vein endothelial cells (HUVEC) following stimulation with phorbol myristate acetate (PMA) were studied; Changes in the Rho-associated coiled-coil protein kinase 1 (ROCK1) protein content and activity were evaluated. DEX and fasudil significantly inhibited U937 cell adhesion rates under PMA stimulation and inhibited ROCK1 activity. Mifepristone (RU-486) and cycloheximide (CHX) did not alter these effects of DEX. DEX interferes with the adhesion function of U937 cells through the inhibition of ROCK1 activity.

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