Keywords: Insomniac Protein, Isothermal Titration Calorimetry, Molecular Dynamics, Protein-Protein Interactions, Cullin, K Potassium Channel Tetramerization Domain Containing Protein, Ubiquitin Protein Ligase E3, Unclassified Drug, Article, Dissociation Constant, Molecular Model, Molecular Recognition, Priority Journal, Protein Binding, Protein Expression, Protein Protein Interaction, Protein Purification, Static Electricity, Stoichiometry, Ubiquitination, Amino Acid Sequence, Cullin Proteins, Humans, Molecular Dynamics Simulation, Molecular Sequence Data, Protein Conformation, Sequence Homology, Substrate Specificity, Cullin Proteins Chemistry Metabolism, Potassium Channels Chemistry Metabolism, Ubiquitin Metabolism, Protein?rotein Interactions,
Affiliations: *** IBB - CNR ***
Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy
Second University of Napoli, 81100 Caserta, Italy
Institute of Crystallography, C.N.R., 70126 Bari, Italy
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Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations
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. (C) 2014 Elsevier B.V. All rights reserved.
Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations