The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications
- McGill University, Canada
- Kyoto University, Japan
Abstract
Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub involved tubulin post-translational modification. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.
Data availability
Cryo-EM maps have been deposited in EM data bank (EMDB) with accession numbers of EMD-20855 (48-nm averaged Chlamydomonas doublet), EMD-20858 (16-nm averaged Chlamydomonas IJ region) and EMD-20856 (16-nm averaged Tetrahymena IJ region). The model of IJ of Chlamydomonas is available in Protein Data Bank (PDB) with an accession number of PDB: 6VE7.The mass spectrometry is deposited in DataDryad (doi:10.5061/dryad.d51c59zxt). Available at:https://datadryad.org/stash/share/bkrXp5Ww0iQUis6ocuEya2ivHWQ_YiTFO-VLeIjkQcM
-
48-nm repeat unit of the doublet microtubule from Chlamydomonas reinhardtiiElectron Microscopy Data Bank, EMD-20855.
-
16-nm averaged Chlamydomonas IJ regionElectron Microscopy Data Bank, EMD-20858.
-
16-nm repeat of the doublet microtubule from Tetrahymena thermophilaElectron Microscopy Data Bank, EMD-20856.
Article and author information
Author details
Funding
Canadian Institutes of Health Research (PJT-156354)
- Khanh Huy Bui
Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-04954)
- Khanh Huy Bui
Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-04813)
- Javier Vargas
Canada Institute For Advanced Research (Arzieli Global Scholar Program)
- Khanh Huy Bui
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Khalifa 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.
Metrics
-
- 3,960
- views
-
- 555
- downloads
-
- 1,425
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications
eLife 9:e52760.
https://doi.org/10.7554/eLife.52760
Further reading
-
- Structural Biology and Molecular Biophysics
Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.
-
- Structural Biology and Molecular Biophysics
The structural basis for the pharmacology of human G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra- and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V2. Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and Gs-bound state, and the guanosine diphosphate (GDP) release from Gs. To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.
