Biophysical and Structural Characterization of the Centriolar Protein Cep104 Interaction Network

Lenka Rezabkova,Sebastian H.W Kraatz,Anna Akhmanova,Michel O Steinmetz,Richard A Kammerer

doi:10.1074/jbc.m116.739771

Lenka Rezabkova, Sebastian H.W Kraatz + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m116.739771

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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.

Highlights

Cilia and flagella are evolutionary conserved organelles that contain a microtubule (MT)3-based cytoskeleton called the axoneme
Understanding the structure, function, and interaction networks of ciliary proteins is key for a better understanding of these severe diseases and ciliogenesis in general
We characterized in detail the domain organization and interaction network of Cep104, a protein that plays a crucial role in ciliogenesis

Summary

Results

Biophysical Characterization of the Cep104 Domains—To better understand the role of Cep104 in ciliogenesis, we first analyzed its amino acid sequence by secondary structure prediction and protein threading that revealed the presence of four conserved functional domains (Fig. 1A). C(S) distribution revealed a single peak with a sw value of 2.5 that amounts to a Smax/S of 1.36 (theoretical molecular mass of monomer: 29 kDa) This result suggests a moderately elongated monomeric structure in solution, which is characteristic of TOG domains. To confirm a direct interaction between the C-terminal part of the Cep104 and the EB homology domain of EB1 proteins and to assess the binding affinities, we performed SV AUC measurements For these experiments, we used the C-terminal part of Cep104 that consists of the zinc-finger domain followed by a 40-amino acid long unstructured region. By measuring different protein concentration ratios using SV AUC, we determined that the C-terminal part of Cep104 directly interacts with the EB homology domain dimers with an apparent dissociation Kd of 2 ␮M (Fig. 4D). The strength of the interaction is comparable with those of other known EB-binding proteins

Discussion

Experimental Procedures

Data collection

TOG native dataset