Structure-based design of a pathway-specific nuclear import inhibitor

Abstract

Kapbeta2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx((2-5))PY motif. Here we show that Kapbeta2 complexes containing hydrophobic and basic PY-NLSs, as classified by the composition of an additional N-terminal motif, converge in structure only at consensus motifs, which explains ligand diversity. On the basis of these data and complementary biochemical analyses, we designed a Kapbeta2-specific nuclear import inhibitor, M9M.

Figure 1

Kapβ2 bound to bPY-NLS of hnRNP M. (a) Ribbon model of Kapβ2 (pink), hnRNP M NLS (magenta) and the 2.5 σ F_o – F_c map (blue). (b) NLSs of hnRNP M (magenta) and hnRNP A1 (2H4M; blue) upon superposition of Kapβ2 residues 435–780. Regions of structural similarity are highlighted in yellow. Structurally aligned NLS sequences, Cα–Cα distances and inhibitor M9M sequence are shown. (c) Loss of Kapβ2-binding energy in alanine mutants of hnRNP A1 (ref. 2) and hnRNP M (ΔΔG = –RTln(K_d(WT)/K_d(mutant)); K_ds determined by ITC).

Figure 2

M9M in vitro and in vivo inhibition studies. (a–c) Coomassie-stained gels of (a) glutathione S-transferase (GST) fusions of hnRNP A1 NLS, hnRNP M NLS and M9M bound to Kapβ2 and then dissociated by 0.3–1.6 μM RanGTP; (b) GST–hnRNP A1 NLS bound to Kapβ2 in the presence of buffer, maltose-binding protein (MBP)–hnRNP A1 NLS, MBP–hnRNP M NLS or MBP-M9M; (c) interactions of GST-Kapβ1 with Kapα, Kapα in the presence of importin-β–binding (IBB) domain of Kapα, M9M or Kapα in the presence of M9M. (d,e) Immunofluorescence and deconvolution microscopy of HeLa cells transfected with plasmids encoding Myc-tagged MBP or MBP-M9M, using anti-Myc and antibodies to hnRNP A1, hnRNP M and HuR. Histogram shows percentages of transfected cells with cytoplasmic Kapβ2 substrates. (f) Same as e, except localization of endogenous HDAC1 (Kapα–Kapβ1 substrate) is determined as control.