Biophysical and Structural Characterization of the Centriolar Protein Cep104 Interaction Network

Abstract

Dysfunction of cilia is associated with common genetic disorders termed ciliopathies. Knowledge on the interaction networks of ciliary proteins is therefore key for understanding the processes that are underlying these severe diseases and the mechanisms of ciliogenesis in general. Cep104 has recently been identified as a key player in the regulation of cilia formation. Using a combination of sequence analysis, biophysics, and x-ray crystallography, we obtained new insights into the domain architecture and interaction network of the Cep104 protein. We solved the crystal structure of the tumor overexpressed gene (TOG) domain, identified Cep104 as a novel tubulin-binding protein, and biophysically characterized the interaction of Cep104 with CP110, Cep97, end-binding (EB) protein, and tubulin. Our results represent a solid platform for the further investigation of the microtubule-EB-Cep104-tubulin-CP110-Cep97 network of proteins. Ultimately, such studies should be of importance for understanding the process of cilia formation and the mechanisms underlying different ciliopathies.

Keywords: Cep104; TOG domain; biophysics; centriole; cilia; circular dichroism (CD); x-ray crystallography.

FIGURE 1.

Biophysical characterization of Cep104 domains. A, schematic representation of the Cep104 interaction network. Domains are shown as cylinders, jelly-roll in black, coiled coil in red, TOG in blue, and zinc finger in green. Interactions are denoted by arrows; B, CD spectra of Cep104 domains. The spectra were recorded using a protein concentration of 5 μm. C, c(S) distributions show a single peak for all Cep104 domains. The jelly-roll, TOG, and zinc-finger domains are monomeric, whereas the coiled coil is dimeric.

FIGURE 2.

Cep104 is a novel tubulin binding protein. A, s_w isotherm showing the interaction between the TOG domain of Cep104 and tubulin. B, SV AUC profiles showing the interaction between the TOG domain of Cep104 (residues 418–673) and tubulin. Brown curve, tubulin; blue curve, mixture of 1 μm tubulin and 0.5 μm TOG domain; cyan curve, mixture of 1 μm tubulin and 1 μm TOG domain; magenta, mixture of 1 μm tubulin and 2 μm TOG domain; green curve, mixture of 1 μm tubulin and 5 μm TOG domain; C and D, s_w isotherms showing that mutation of Trp-448 or Arg-626 completely abolishes binding to tubulin; E, schematic representation of x-ray structure of the Cep104 TOG domain comprising 6 HEAT repeats (HR1–6) and 5 loop regions (T1-T5). The loop regions form the tubulin-binding site; F, model of a complex between the TOG domain of Cep104 and an unpolymerized tubulin dimer in the curved conformation that was generated based on the x-ray crystal structure of the TOG1 domain of the Stu2 complexed with yeast tubulin dimer (PDB code 4FFB). The model was generated by superimposition of the Cep104 TOG domain with the TOG1 domain of the Stu2-TOG1-tubulin complex. Residues important for the interaction with tubulin, Trp-448 and Arg-626 in Cep104 TOG, and Trp-23 and Arg-200 in Stu2-TOG1, are highlighted with a red circle. Blue, α-tubulin; orange, β-tubulin; gray, Cep104 TOG domain; black, Stu2-TOG1; G, surface charge representation of the x-ray crystal structure of the Cep104 TOG domain colored according to its electrostatic potential (±8 kT/e, where k is the Boltzmann constant, T is temperature, and e is the elementary charge): blue denotes basic residues, red denotes acidic residues, and white denotes hydrophobic residues. Residues important for the interaction with tubulin are labeled and highlighted with a red circle.

FIGURE 3.

Mapping of the interaction between the C-terminal part of Cep104 (residues 730–925) and different CP110 polypeptide chain fragments. A, schematic representation of CP110 polypeptide chain fragments used in this study; B-G, SV AUC profiles of the C-terminal part of Cep104 (magenta), different uncleaved thioredoxin-CP110 fusion proteins (residues 902–991, 926–991, 902–946, 912–936, 902–925, 907–936 in green) and their mixtures (blue). Only CP110 polypeptide chain fragments containing residues 902–991, 902–946, and 907–936 have the ability to interact with Cep104. The experiments were performed using a protein concentration of 30 μm.

FIGURE 4.

Cep104 forms a dimer in solution that directly interacts with CP110, Cep97, and EB. A, ITC measurement of the interaction between the C-terminal part of Cep104 (residues 730–925) and the synthetic CP110 peptide (residues 907–936). The CP110 peptide interacts with Cep104 with an apparent dissociation constant of 4 μm and in a 1:1 stoichiometry; B, normalized thermal unfolding profile and of the Cep104 coiled-coil region (residues 207–301); C, SV AUC profiles showing the interaction between the N-terminal part of Cep104 (residues 1–163) and central part of Cep97 (residues 310–480). Green, Cep97; magenta, Cep104; blue, mixture of 5 μm Cep97 and 5 μm Cep104; cyan, mixture of 10 μm Cep97 and 10 μm Cep104; red, mixture of 15 μm Cep97 and 15 μm Cep104; black, mixture of 20 μm Cep97 and 20 μm Cep104; D, SV AUC profiles showing the interaction between the C-terminal part of Cep104 (residues 730–925) and EB1 (residues 191–267). Green, EB1; magenta, Cep104; brown, mixture of 1 μm EB and 1 μm Cep104; blue, mixture of 2 μm EB and 2 μm Cep104; cyan, mixture of 5 μm EB and 5 μm Cep104; red, mixture of 10 μm EB and 10 μm Cep104; black, mixture of 15 μm EB and 15 μm Cep104.