The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor's ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.

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A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health

Science Advances ◽

10.1126/sciadv.abf1002 ◽

2021 ◽

Vol 7 (18) ◽

pp. eabf1002

Author(s):

Aileen J. Lam ◽

Lu Rao ◽

Yuzu Anazawa ◽

Kyoko Okada ◽

Kyoko Chiba ◽

...

Keyword(s):

Molecular Basis ◽

Fundamental Principle ◽

Association Rate ◽

Biophysical Parameters ◽

Kinesin Motor ◽

Cargo Transport ◽

The Family ◽

Loop Conformation ◽

Conserved Residue ◽

310 Helix

KIF1A is a critical cargo transport motor within neurons. More than 100 known mutations result in KIF1A-associated neurological disorder (KAND), a degenerative condition for which there is no cure. A missense mutation, P305L, was identified in children diagnosed with KAND, but the molecular basis for the disease is unknown. We find that this conserved residue is part of an unusual 310 helix immediately adjacent to the family-specific K-loop, which facilitates a high microtubule-association rate. We find that the mutation negatively affects several biophysical parameters of the motor. However, the microtubule-association rate of the motor is most markedly affected, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that the 310 helix facilitates a specific K-loop conformation that is critical for its function. We find that the function of this proline is conserved in kinesin-1, revealing a fundamental principle of the kinesin motor mechanism.

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Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31692-2 ◽

1994 ◽

Vol 269 (51) ◽

pp. 32708

Author(s):

T G Huang ◽

J Suhan ◽

D D Hackney

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Position ◽

Motor Domain ◽

Kinesin Motor

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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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Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli

Biochemistry ◽

10.1021/bi00068a028 ◽

1993 ◽

Vol 32 (17) ◽

pp. 4677-4684 ◽

Cited By ~ 52

Author(s):

Susan P. Gilbert ◽

Kenneth A. Johnson

Keyword(s):

Escherichia Coli ◽

Motor Domain ◽

Purification And Characterization ◽

Kinesin Motor

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Analyzing kinesin motor domain translocation in cultured hippocampal neurons

Methods in Cell Biology - The Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon ◽

10.1016/bs.mcb.2015.06.021 ◽

2016 ◽

pp. 217-232 ◽

Cited By ~ 4

Author(s):

Rui Yang ◽

Marvin Bentley ◽

Chung-Fang Huang ◽

Gary Banker

Keyword(s):

Hippocampal Neurons ◽

Motor Domain ◽

Kinesin Motor ◽

Cultured Hippocampal Neurons

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Function of the p97–Ufd1–Npl4 complex in retrotranslocation from the ER to the cytosol

The Journal of Cell Biology ◽

10.1083/jcb.200302169 ◽

2003 ◽

Vol 162 (1) ◽

pp. 71-84 ◽

Cited By ~ 439

Author(s):

Yihong Ye ◽

Hemmo H. Meyer ◽

Tom A. Rapoport

Keyword(s):

Atp Hydrolysis ◽

Bound State ◽

Polyubiquitin Chain ◽

Polyubiquitin Chains ◽

Evolutionarily Conserved ◽

The Family ◽

Complex Functions ◽

Ubiquitin Binding ◽

Subsequent Degradation ◽

Er Membrane

Amember of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1–Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97–Ufd1–Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

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Kinetic Mechanism of Kinesin Motor Domain

Biochemistry ◽

10.1021/bi00040a039 ◽

1995 ◽

Vol 34 (40) ◽

pp. 13233-13241 ◽

Cited By ~ 49

Author(s):

Y. Z. Ma ◽

Edwin W. Taylor

Keyword(s):

Kinetic Mechanism ◽

Motor Domain ◽

Kinesin Motor

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A unique kinesin-8 surface loop provides specificity for chromosome alignment

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1132 ◽

2014 ◽

Vol 25 (21) ◽

pp. 3319-3329 ◽

Cited By ~ 26

Author(s):

Haein Kim ◽

Cindy Fonseca ◽

Jason Stumpff

Keyword(s):

Motor Domain ◽

Common Mechanism ◽

Surface Loop ◽

C Terminus ◽

Chromosome Alignment ◽

Spindle Microtubules ◽

And Function ◽

Positively Charged ◽

Mitotic Chromatin

Microtubule length control is essential for the assembly and function of the mitotic spindle. Kinesin-like motor proteins that directly attenuate microtubule dynamics make key contributions to this control, but the specificity of these motors for different subpopulations of spindle microtubules is not understood. Kif18A (kinesin-8) localizes to the plus ends of the relatively slowly growing kinetochore fibers (K-fibers) and attenuates their dynamics, whereas Kif4A (kinesin-4) localizes to mitotic chromatin and suppresses the growth of highly dynamic, nonkinetochore microtubules. Although Kif18A and Kif4A similarly suppress microtubule growth in vitro, it remains unclear whether microtubule-attenuating motors control the lengths of K-fibers and nonkinetochore microtubules through a common mechanism. To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A. Each of these chimeric kinesins localizes to K-fibers; however, K-fiber length control requires an activity specific to kinesin-8s. Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain. These data support a model in which microtubule-attenuating kinesins are molecularly “tuned” to control the dynamics of specific subsets of spindle microtubules.

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Kinesin Motor Domain of Leishmania donovani as a Future Vaccine Candidate

Clinical and Vaccine Immunology ◽

10.1128/cvi.00433-07 ◽

2008 ◽

Vol 15 (5) ◽

pp. 836-842 ◽

Cited By ~ 12

Author(s):

Ayan Dey ◽

Pawan Sharma ◽

Naresh Singh Redhu ◽

Sarman Singh

Keyword(s):

Visceral Leishmaniasis ◽

Lymphocyte Proliferation ◽

Leishmania Donovani ◽

Mononuclear Cells ◽

Vaccine Candidate ◽

Ex Vivo ◽

Interleukin 2 ◽

Motor Domain ◽

Enzyme Linked Immunosorbent Assay ◽

Kinesin Motor

ABSTRACT Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.

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