In silico characterization of the mechanisms of interaction between nuclear localization sequences and Importin-α

Author(s)

Marcos Tadeu Geraldo

• Document type: Doctoral Thesis
• Press: Botucatu. 2016-08-15.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2016-07-26
• Advisor: Ney Lemke; Agnes Alessandra Sekijima Takeda

Abstract

Nuclear import systems are responsible for the exchange between the cytoplasm and the nucleus of a cell, allowing nuclear proteins to migrate through the membrane that separates these two regions. The most studied import pathway is the classical nuclear import mediated by Importin-α (Impα). Impα is a solenoid protein consisting of tandem repeats of the Armadillo (ARM) motif, forming an extended and twisted structure with small grooves along the protein axis. The classical nuclear localization sequences (cNLSs) of cargo proteins are composed of positively charged residues and establish salt bridges, hydrogen bonds and hydrophobic contacts with the grooves of Impα. Such recognition can occur at one or two sites of Impα, thus characterizing the cNLS as monopartite or bipartite, respectively. Most structural information of the cNLS-Impα complex is from crystallographic data and little is known about the conformational dynamics of this system. One approach to address the dynamics of a system is the use of biomolecular simulation techniques such as molecular dynamics and normal modes analysis. Based on these techniques, this study aimed to understand the mechanisms of interaction and conformational dynamics involved in the recognition of cNLSs by Impα. In particular, this work focused on the cNLSs of Nucleoplasmin and Ku70 proteins complexed with Impα. The study of Nucleoplasmin determined two main motions of Impα that may be associated to the cNLS recognition: bending and twisting. The bending motion may be involved in the cNLS entrance and the accommodation of cargo protein depending on its size, whereas the twisting motions may be involved in the cNLS recognition and accommodation into the binding sites of Impα. Furthermore, the residues corresponding to the linker region, situated between the groups of basic residues from the bipartite cNLS, may also assist in setting the cNLS into Impα. Finally, the study of Ku70 verified, based on the contact and correlation analyses of the peptide-protein interface and the geometric profiling of Impα, that its peptide would not bind to Impα as a bipartite cNLS. In conclusion, the data obtained here may help in understanding the affinities between the cNLSs already described, as well as the analysis of other potential cNLSs. (AU)

• FAPESP's process: 12/19447-2 - Dynamics of the interaction between Ku70NLS and Importin-alpha in the classical nuclear import pathway
• Grantee: Marcos Tadeu Geraldo
• Support Opportunities: Scholarships in Brazil - Doctorate