Abstract
We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.
Highlights
The localization, complexity and variability of DNA damage requires a complex cellular response that often utilizes scaffold proteins to recruit and coordinate the activities of the individual repair enzymes required to correct the damage
In view of these variable results for the X-ray cross complementing group 1 protein (XRCC1) nuclear localization signal (NLS) and the essential requirement of nuclear localization of XRCC1 in order to fulfill its repair functions as a scaffold protein, we have further investigated the structural basis for the nuclear uptake of this protein
For crystallographic structural analysis of the complex formed between the hXRCC1 nuclear localization sequence and murine Importin α (Impα) [70–529], we selected a peptide that encompasses all of the previously proposed XRCC1 NLS binding motifs
Summary
The localization, complexity and variability of DNA damage requires a complex cellular response that often utilizes scaffold proteins to recruit and coordinate the activities of the individual repair enzymes required to correct the damage. Variations of the XRCC1 NLS to the repair protein aprataxin in order to effect nuclear uptake These studies indicated that the minimum segment required for efficient nuclear transport included an additional 10 residues, 239–27615. Computational prediction of potential XRCC1 NLS motifs resulted in the identification of two monopartite sequences corresponding to residues 242–250 and 267–276 These two regions are included in the extended NLS identified by Kiriyama et al., the separation between the two binding motifs is atypically long (Fig. 1). XRCC1 represents a challenging cargo protein for the nuclear import machinery due to its floppy structure that includes two unstructured linker sequences greater than 120 residues in length, as well as its ability to interact with multiple DNA-repair binding partners. XRCC1 has been shown to mediate nuclear translocation of JWA, a microtubule-associated protein involved in activation of MAPK cascades
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