An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

Abstract

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 27,000 XLs, we identified 9,208 unique amino acid interactions among 1,368 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.

The MBO2/FAP58 heterodimer stabilizes assembly of inner arm dynein b and reveals axoneme asymmetries involved in ciliary waveform

“…We propose that MBO2 and FAP58 form a coiled-coil heterodimer that complements the role of a FAP189 homodimer on different DMTs or regions. Recent chemical crosslinking and proteomic analysis of the MBO2 and FAP58 orthologs in Tetrahymena indicate that these two subunits interact closely throughout their lengths in the Tetrahymena axoneme ( McCafferty et al. , 2023 ), consistent with our hypothesis.…”

“…The placement of the MBO2/FAP58 heterodimer is also consistent with the mapped positions of short segments of amino acids that are conserved between FAP189 and FAP58 (See Figure Legend 7 and Walton et al. , 2023 ) and recent chemical cross-linking experiments suggesting the presence of a CCDC146/CCDC147 heterodimer in Tetrahymena axonemes ( McCafferty et al. , 2023 ).…”

DomainFit: Identification of Protein Domains in cryo-EM maps at Intermediate Resolution using AlphaFold2-predicted Models

“…Mass spectrometry results show that CCDC81B is resistant to salt and is similar to a MIP profile 21. In situ cross-linking mass spectrometry shows that CCDC81B is crosslinked to tubulin alpha, CFAP106, FAP210, and interestingly RIB43A 36. All these cross-links are satisfied relative to the CCDC81B position in the unknown density, except for RIB43A (Fig.…”

“…In addition, BMIP1 is abundant in both salt-treated and native doublet microtubules similar to the profiles of most MIPs. BMIP1 has a cross-link to CFAP45, which exists as two copies between protofilaments B8B9 and B7B8, as shown in studies that usedchemical cross-linking coupled with mass spectrometry of cilia 21,36 (Fig. 6C).…”

“…For the modelling of CCDC81B (UniprotID I7M688, TTHERM_00649260), best fitting positions of different domains of CCDC81B were found using DomainFit. We joined all the domains together using Coot 47 and refined in Phenix 36.…”