Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

Abstract

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.

Fig. 1. Structure and comparative analysis of ^Nab2PY-NLS

(A) Sequence alignment of several PY-NLSs. This signal consists of an N-terminal hydrophobic or basic motif (green and blue, respectively) and a C-terminal RX_2-5PY motif (red). (B) Overall structure of the Kapβ2•^Nab2PY-NLS complex. The karyopherin is in pink and the PY-NLS cyan. (C) mFo-DFc difference map, contoured at 2.0σ (grey), at the PY-NLS binding site of Kapβ2 before the ^Nab2PY-NLS residues were included in the model. The final refined model of ^Nab2PY-NLS residues Thr234 to Ala240 are in cyan. (D) Superposition of Kapβ2 residues 301-634 for Kapβ2s bound to PY-NLSs from cargos Nab2 and hnRNP A1 (PDB ID 2H4M; Cα rmsd 0.35 Å). Kapβ2 of the Nab2 complex is shown in pink. The Nab2 PY-NLS is in cyan and the hnRNP A1 PY-NLS is in yellow. (E) Similar superpositions of Kapβ2 as in D, to compare PY-NLSs from Nab2 (cyan), hnRNP A1 (yellow), hnRNP M (green), hnRNP D (orange), and TAP/NXF1 (purple).

Fig. 2. The Kapβ2•^Nab2PY-NLS Interface

(A) The ^Nab2PY-NLS (cyan) makes numerous hydrophobic contacts with Kapβ2 (pink). In addition, Arg-235 of the ^Nab2PY-NLS makes multiple salt bridges and hydrogen bonds with Kapβ2. Interacting residues on Kapβ2 are labeled in black and contacts (4.0 Å or less) are indicated by dashed lines. (B) Comparison of the PY motifs of PY-NLSs from Nab2 (cyan) and hnRNP D (orange). Intramolecular contacts (4.0 Å or less) in hnRNP D are shown with dashed lines.