A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health.
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ABSTRACT:
SUBMITTER: Lam AJ
PROVIDER: S-EPMC8087401 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.
| S-EPMC3820893 | biostudies-literature
Project description:More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34-Å resolution X-ray crystallographic structure of a previously uncharacterized 'domain of unknown function' 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologues from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.
| S-EPMC4352575 | biostudies-literature
Project description:An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.
| S-EPMC3562449 | biostudies-literature
Project description:Kinesin is a motor protein essential in cellular functions, such as intracellular transport and cell-division, as well as for enabling nanoscopic transport in bio-nanotechnology. Therefore, for effective control of function for nanotechnological applications, it is important to be able to modify the function of kinesin. To circumvent the limitations of chemical modifications, here we identify another potential approach for kinesin control: the use of electric forces. Using full-atom molecular dynamics simulations (247,358 atoms, total time ∼ 4.4 μs), we demonstrate, for the first time, that the kinesin-1 motor domain can be detached from a microtubule by an intense electric field within the nanosecond timescale. We show that this effect is field-direction dependent and field-strength dependent. A detailed analysis of the electric forces and the work carried out by electric field acting on the microtubule-kinesin system shows that it is the combined action of the electric field pulling on the β-tubulin C-terminus and the electric-field-induced torque on the kinesin dipole moment that causes kinesin detachment from the microtubule. It is shown, for the first time in a mechanistic manner, that an electric field can dramatically affect molecular interactions in a heterologous functional protein assembly. Our results contribute to understanding of electromagnetic field-biomatter interactions on a molecular level, with potential biomedical and bio-nanotechnological applications for harnessing control of protein nanomotors.
| S-EPMC9939557 | biostudies-literature
Project description:Kinesin superfamily members play important roles in many diverse cellular processes, including cell motility, cell division, intracellular transport, and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their different cellular roles remains largely unknown. Here, we employed molecular-dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. We then used experimental mutagenesis and single-molecule motility assays to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11, and α6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely, R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.
| S-EPMC4624112 | biostudies-literature
Project description:Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome-MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.
| S-EPMC2063730 | biostudies-literature
Project description:Introduction:Kinesin-1 motor is a molecular walking machine constructed with amino acids. The understanding of how those structural elements play their mechanical roles is the key to the understanding of kinesin-1 mechanism. Methods:Using molecular dynamics simulations, we investigate the role of a helix structure, ?4 (also called switch-II helix), of kinesin-1's motor domain in its processive movement along microtubule. Results:Through the analysis of the structure and the interactions between ?4 and the surrounding residues in different nucleotide-binding states, we find that, mechanically, this helix functions as a shaft for kinesin-1's motor-domain rotation and, structurally, it is an amphipathic helix ensuring its shaft functioning. The hydrophobic side of ?4 consists strictly of hydrophobic residues, making it behave like a lubricated surface in contact with the core ?-sheet of kinesin-1's motor domain. The opposite hydrophilic side of ?4 leans firmly against microtubule with charged residues locating at both ends to facilitate its positioning onto the intra-tubulin groove. Conclusions:The special structural feature of ?4 makes for an effective reduction of the conformational work in kinesin-1's force generation process.
| S-EPMC6816713 | biostudies-literature
Project description:Members of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization.This article has an associated First Person interview with the first author of the paper.
| S-EPMC7340583 | biostudies-literature
Project description:Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the alpha-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin-MT interface without changing important structural features within the motor's core. Finally, we identify casein kinase 1alpha as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization.
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Project description: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.
| S-EPMC2394829 | biostudies-literature