Distinctive Conformation of Minor Site-Specific Nuclear Localization Signals Bound to Importin-[alpha]

Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin-[alpha]. There are two NLS-binding sites (major and minor) on importin-[alpha] and the major NLS-binding site is considered to be the primary binding site. Here, we used crystallographic and biochemical methods to investigate the binding between importin-[alpha] and predicted 'minor site-specific' NLSs: four peptide library-derived peptides, and the NLS from mouse RNA helicase II/Gu[alpha]. The crystal structures reveal that these atypical NLSs indeed preferentially bind to the minor NLS-binding site. Unlike previously characterized NLSs, the C-terminal residues of these NLSs form an [alpha]-helical turn, stabilized by internal H-bond and cation-π interactions between the aromatic residues from the NLSs and the positively charged residues from importin-[alpha]. This helical turn sterically hinders binding at the major NLS-binding site, explaining the minor-site preference. Our data suggest the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal minor NLS-binding site-specific motif, which may help identify novel proteins with atypical NLSs.[Image omitted see PDF] [PUBLICATION ABSTRACT]