Distinctive Conformation of Minor Site‐Specific Nuclear Localization Signals Bound to Importin‐α

Nuclear localization signals (NLSs) contain one or two clusters of basic residues that are recognized by importin‐α. Importin‐α has two NLS‐binding sites (major and minor), with the major site known as the primary binding site. Using crystallographic and biochemical approaches, we have characterized a group of atypical NLSs that bind primarily to the minor site. Unlike previously characterized NLSs, the C‐terminal residues of these NLSs form an α‐helical turn, which prevents them from binding to the major NLS‐binding site. Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin‐α. There are two NLS‐binding sites (major and minor) on importin‐α 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‐α and predicted ‘minor site‐specific’ NLSs: four peptide library‐derived peptides, and the NLS from mouse RNA helicase II/Guα. 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 α‐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‐α. 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.