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

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• Cilia are vital eukaryotic organelles involved in motility, signaling, and sensory reception.
• Intraflagellar transport (IFT) is crucial for cilium formation, utilizing IFT-A and IFT-B complexes transported by molecular motors.
• The IFT-B complex forms the backbone of IFT trains, yet high-resolution structures are lacking for over 50% of its components.

Purpose of the Study:

• To determine the high-resolution structure of the complete 15-subunit IFT-B complex.
• To provide a structural framework for understanding IFT-B assembly, function, and its role in ciliopathies.
• To investigate potential variations in IFT-B complex architecture.

Main Methods:

• Computational structural modeling using AlphaFold.
• Validation through cross-linking/mass spectrometry (XL-MS) on reconstituted complexes.
• Experimental validation including X-ray scattering, crystallography, site-directed mutagenesis, and protein-binding assays.

Main Results:

• A validated, high-resolution structural model of the entire 15-subunit IFT-B complex was generated.
• The IFT-B structure is elongated and flexible, aligning with cryo-electron tomographic data of IFT trains.
• The complex comprises IFT-B1 and IFT-B2 regions, with distinct binding sites for ciliary cargo and the IFT dynein motor, respectively.
• Evidence suggests multiple binding sites for specific subunits (IFT81/74) on others (IFT88/70/52/46), indicating potential structural plasticity in the IFT-B1 subcomplex.

Conclusions:

• The study presents the first comprehensive structural model of the IFT-B complex.
• This structural framework is essential for understanding IFT-B function, assembly dynamics, and the molecular basis of ciliopathies.
• The findings highlight the structural flexibility of the IFT-B complex and its implications for cargo transport and motor interactions.