title: Molecular recognition of Cullin3 by KCTDs: insights from experimental and computational investigations.
abstract: Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5-Cul3)(5) pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy; Second University of Napoli, 81100 Caserta, Italy.
authors.forename: Nicole
authors.initials: N
authors.lastname: Balasco
authors.affiliation: Institute of Crystallography, C.N.R., 70126 Bari, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Pirone
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Smaldone
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Di Gaetano
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Esposito
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.
authors.forename: Emilia Maria
authors.initials: EM
authors.lastname: Pedone
authors.affiliation: Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy. Electronic address: luigi.vitagliano@unina.it.
authors.forename: Luigi
authors.initials: L
authors.lastname: Vitagliano
mesh_terms: D000595:Amino Acid Sequence; D044842:Cullin Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C503645:CUL3 protein, human; D044842:Cullin Proteins; C521187:KCTD5 protein, human; D015221:Potassium Channels; D025801:Ubiquitin
keywords: Insomniac protein; Isothermal titration calorimetry; Molecular dynamics; Protein–protein interactions