scholarly journals The mechanism of selective kinesin inhibition by kinesin binding protein

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.

scholarly journals The mechanism of kinesin inhibition by kinesin-binding protein

eLife ◽  
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.

scholarly journals The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Joseph Atherton ◽  
I-Mei Yu ◽  
Alexander Cook ◽  
Joseph M Muretta ◽  
Agnel Joseph ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Motor Function ◽  
Nucleotide Binding ◽  
Motor Domain ◽  
Kinesin Motor ◽  
Binding Motifs ◽  
Cryo Electron Microscopy ◽  
Specific Sequences

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.

scholarly journals Dynamic oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis

2019 ◽  
Author(s):  
Mariusz Madej ◽  
Joshua B. R. White ◽  
Zuzanna Nowakowska ◽  
Shaun Rawson ◽  
Carsten Scavenius ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Direct Evidence ◽  
Inflammatory Diseases ◽  
Extracellular Proteins ◽  
Substrate Selectivity ◽  
Binding Studies ◽  
Uptake Mechanism ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Binding Surface

AbstractPorphyromonas gingivalis, an asaccharolytic Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases, such as rheumatoid arthritis, cardiovascular disease and Alzheimer’s disease. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how those peptides enter the cell is not clear. Here we identify RagAB as the outer membrane importer for peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex with the RagB substrate binding surface-anchored lipoprotein forming a closed lid on the TonB-dependent transporter RagA. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a “pedal bin” nutrient uptake mechanism. Together with mutagenesis, peptide binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic OM oligopeptide acquisition machine with considerable substrate selectivity that is essential for the efficient utilisation of proteinaceous nutrients by P. gingivalis.

Kinetic Mechanism of Kinesin Motor Domain

Biochemistry ◽  
1995 ◽  
Vol 34 (40) ◽  
pp. 13233-13241 ◽  
Author(s):  
Y. Z. Ma ◽  
Edwin W. Taylor
Keyword(s):  
Kinetic Mechanism ◽  
Motor Domain ◽  
Kinesin Motor

scholarly journals Interaction of mammalian neprilysin with binding protein and calnexin in Schizosaccharomyces pombe

1999 ◽  
Vol 340 (3) ◽  
pp. 813-819 ◽  
Author(s):  
Hugues BEAULIEU ◽  
Aram ELAGÖZ ◽  
Philippe CRINE ◽  
Luis A. ROKEACH
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Protein ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Eukaryotic Cell ◽  
Unfolded Proteins ◽  
Folding Intermediates ◽  
Bona Fide ◽  
The Brain

Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.11) is a zinc metallo-endopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles. In the brain (and central nervous system), this enzyme is involved in the molecular mechanism of pain by its action in the degradation of enkephalin molecules. In the kidney, NEP is implicated in the degradation of regulatory factors involved in the control of arterial pressure, including atrial natriuretic peptide and bradykinin. In this study we assessed the potential of the fission yeast Schizosaccharomyces pombe to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein). Both recombinant NEP and sNEP were produced at high levels (5 mg/l) in this system. Enzymic studies revealed that these recombinant proteins were fully active and exhibit kinetic parameters similar to those of the bona fide enzyme. Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to the cell membrane. Furthermore, co-immunoprecipitation studies showed that folding intermediates of NEP and sNEP, produced in S. pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnx1p). The amount of sNEP coprecipitated with both BiP and Cnx1p augmented when cells were subjected to various stresses causing the accumulation of unfolded proteins in the ER. The interactions of NEP with BiP and Cnx1p were, however, more refractive to the same stresses.

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