The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-α (Impα), we co-crystallized non-autoinhibited Impα with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impα. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impα. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impα, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impα.
- importin-α (karyopherin-α)
- nuclear localization sequence recognition (NLS recognition)
- simian-virus-40 (SV40) large tumour-antigen nuclear localization sequence
- X-ray crystal structure
The co-ordinates of pCN and CN peptide complexes have been deposited in the Protein Data Bank with the accession codes 1Q1S and 1Q1T respectively.
Abbreviations used: AcN, acetonitrile; Arm repeat, armadillo repeat; T-antigen, tumour antigen; T-Ag, simian-virus-40 large T-antigen; CN peptide, GPGSDDEAAADAQHAAPPKKKRKVG (corresponding to residues 109–133 of T-Ag with modifications); Imp, importin; IBB domain, Impβ-binding domain; fl-Impα, full-length Impα; NLS, nuclear localization sequence; PAH, phenylalanine hydroxylase; TFA, trifluoroacetic acid.
- The Biochemical Society, London ©2003