Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex
- The University of Texas at Austin, United States
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Human Technopole, Italy
Abstract
Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.
Data availability
Mass spectrometry proteomics data was deposited in the MassIVE/ProteomeXchange database (113) under accession number PXD032818. Cryo-tomography data was deposited in the Electron Microscopy Data Bank (114) under accession number EMD-26791. IFT-A models were deposited in the PDB-Dev database (115) as well as on Zenodo at doi: 10.5281/zenodo.7222413, along with additional supporting materials, including integrative modeling data and code.
Article and author information
Author details
Funding
National Science Foundation (2019238253)
- Caitlyn L McCafferty
National Institute of General Medical Sciences (R35GM122480)
- Edward M Marcotte
National Institute of General Medical Sciences (R35GM138348)
- David W Taylor
National Institute of Child Health and Human Development (HD085901)
- John B Wallingford
- Edward M Marcotte
Army Research Office (W911NF-12-1-0390)
- Edward M Marcotte
Welch Foundation (F-1515)
- Edward M Marcotte
Welch Foundation (F-1938)
- David W Taylor
Max Planck Society
- Mareike A Jordan
- Gaia Pigino
Cancer Prevention and Research Institute of Texas (RR160088)
- David W Taylor
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, McCafferty 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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Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex
eLife 11:e81977.
https://doi.org/10.7554/eLife.81977
Further reading
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Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.
