The mechanism of kinesin inhibition by kinesin binding protein
- King's College London, United Kingdom
- Utrecht University, Netherlands
- Paul Scherrer Institute, Switzerland
- The Francis Crick Institute, United Kingdom
- Pharmidex 19 Pharmaceuticals, United Kingdom
- Mayo Clinic, United States
- Institute of Structural and Molecular Biology, Birkbeck College, United Kingdom
Abstract
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 TPR-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.
Data availability
Cryo-EM electron density maps and models have been deposited in the electron microscopy data bank (EMDB) and protein data bank (PDB) respectively. The relevant deposition codes are provided in Table 1.
Article and author information
Author details
Casper C Hoogenraad
Funding
Medical Research Council (MR/R000352/1)
- Joseph Atherton
Worldwide Cancer Research (16-0037)
- Julia Locke
- Alejandro Peña
Wellcome Trust (202679/Z/16/Z,206166/Z/17/Z and 079605/Z/06/Z)
- Joseph Atherton
- Julia Locke
- Alejandro Peña
Biotechnology and Biological Sciences Research Council (BB/L014211/1)
- Joseph Atherton
- Julia Locke
- Alejandro Peña
National Institute of General Medical Sciences (R01GM130556)
- Steven S Rosenfeld
Swiss National Science Foundation (31003A_166608)
- Natacha Olieric
- Michel O Steinmetz
Netherlands Organization for Scientific Research (NWO-ALW-VICI,CCH)
- Jessica JA Hummel
- Casper C Hoogenraad
European Research Council (ERC-consolidator,CCH)
- Jessica JA Hummel
- Casper C Hoogenraad
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Atherton et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The mechanism of kinesin inhibition by kinesin binding protein
eLife 9:e61481.
https://doi.org/10.7554/eLife.61481
Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.
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- Structural Biology and Molecular Biophysics
Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. These observations are corroborated by electrophysiological experiments showing that ion permeation can be resumed in the kv1.2-kv2.1-3m channel when I398 is mutated to an asparagine—a mutation that does not abolish C-type inactivation since digitoxin (AgTxII) fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of quaternary ammonium (QA) blockers and negatively charged activators thus opening new research directions toward the development of drugs that specifically modulate gating states of Kv channels.
