Faculty of Natural Sciences

TY - JOUR

T1 - Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B

AU - Petriman, Narcis A

AU - Loureiro-López, Marta

AU - Taschner, Michael

AU - Zacharia, Nevin K

AU - Georgieva, Magdalena M

AU - Boegholm, Niels

AU - Wang, Jiaolong

AU - Mourão, André

AU - Russell, Robert B

AU - Andersen, Jens S

AU - Lorentzen, Esben

N1 - ©2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

PY - 2022/12

Y1 - 2022/12

N2 - Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

AB - Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

KW - AlphaFold

KW - Cilium

KW - IFT-B structure

KW - Intraflagellar transport

KW - Structural modeling

UR - http://www.scopus.com/inward/record.url?scp=85141948022&partnerID=8YFLogxK

U2 - 10.15252/embj.2022112440

DO - 10.15252/embj.2022112440

M3 - Journal article

C2 - 36354106

VL - 41

JO - E M B O Journal

JF - E M B O Journal

SN - 0261-4189

IS - 24

M1 - e112440

ER -