Structural and biochemical characterization of the recognition of the 53BP1 nuclear localization signal by importin-α

53BP1 (TP53-binding protein 1) plays a key role in DNA double-strand break repair by promoting nonhomologous end joining (NHEJ) especially during G1 phase of the cell cycle. Nuclear import of 53BP1 is required for proper localization of 53BP1 and maintenance of genome integrity. 53BP1 has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686. Ser1678 within the 53BP1 NLS can be phosphorylated by CDK1/cyclin B, and a phosphomimetic substitution of Ser1678 with aspartate has been shown to negatively regulate nuclear import of 53BP1. Here, the X-ray crystal structures of the nuclear import adaptor importin-alpha 1 bound to the wild-type 53BP1 NLS and the S1678D mutant of 53BP1 NLS are reported at resolutions of 1.9 and 1.7 A, respectively. In the wild-type structure, not only the two basic clusters of the 53BP1 NLS but also the linker region between the basic clusters made extensive interactions with importin-alpha 1. In the mutant structure, the linker region between the basic clusters in the 53BP1 NLS made fewer interactions with importin-alpha 1 than those observed in the wild-type structure. However, biochemical binding assays using purified proteins showed that the 53BP1 mutation S1678D reduces the binding affinity to importin-alpha 1 only to a modest extent. Implications of these findings for regulatory mechanism of 53BP1 nuclear import are discussed. (C) 2019 Elsevier Inc. All rights reserved.