Biochemically validated structural model of the 15-subunit IFT-B complex

Abstract

Cilia are ubiquitous eukaryotic organelles important to cellular motility, signalling and sensory reception. Cilium formation requires intraflagellar transport for trafficking of structural and signalling components. The large MDa IFT-B complex constitutes the backbone of polymeric IFT trains that carry ciliary cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B sub-complexes leaving >50% of the IFT-B complex structurally uncharacterized. We have used recent advances in protein structure prediction as implemented in Alphafold to assemble a structural model for the 15-subunit IFT-B complex. The model was validated using crosslinking/MS data on reconstituted IFT-B complexes, X-ray scattering in solution and diffraction from crystals as well as site-directed mutagenesis and protein binding assays. The IFT-B structural model reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The >400[A] long IFT-B complex can roughly be divided into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our structural modelling and crosslinking/MS results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of two different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.