Kapβ2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx (2)(3)(4)(5) PY motif. Here we show that Kapβ2 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 Kapβ2-specific nuclear import inhibitor, M9M.Ten different import karyopherin-βs (Kapβs, also called importin-βs) 1 mediate trafficking of human proteins into the cell nucleus through recognition of distinct NLSs. Large panels of import substrates are known only for Kapβ1 (importin-β) and Kapβ2 (transportin) 1,2 . The substrate repertoire of each Kapβ and the functional consequences of pathway specificities are some of the main challenges in understanding intracellular signaling and trafficking. In the case of nuclear export, Crm1 inhibitor leptomycin B has been crucial for identifying many Crm1 substrates 3,4 . Such specific inhibitors of nuclear import could be invaluable for proteomic analyses to map extensive nuclear traffic, but none has been found. Two classes of NLS are currently known: short, basic classical NLSs that bind the heterodimer 5), and newly identified PY-NLSs that bind Kapβ2 (ref.2). PY-NLSs are 20-to 30-residue signals with intrinsic structural disorder, overall basic character, C-terminal R/K/Hx 2-5 PY motifs (where x 2-5 is any sequence of 2-5 residues) and N-terminal hydrophobic or basic motifs. These weak but orthogonal characteristics have provided substantial limits in sequence space, enabling the identification of over 100 PY-NLS-containing human proteins 2 . Two subclasses, hPY-NLSs and bPY-NLSs, are defined by their N-terminal motifs: hPY-NLSs contain ϕG/A/Sϕϕ motifs (where ϕ is a hydrophobic residue), whereas bPY-NLSs are enriched with basic residues.We have previously solved the structure of human Kapβ2 bound to the hPY-NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) 2 . Here we have solved the 3.1-Å Fig. 1a,b; Kapβ2 435-780 Cα r.m.s. deviation is 0.9Å). Their NLS termini are structurally diverse, consistent with their apparent lack of sequence conservation 2 . At the N terminus, hnRNP A1 residues 263-266 bind the convex side of Kapβ2 (ref.2), whereas the N terminus of hnRNP M proceeds toward the Kapβ2 arch opening. At the C terminus, hnRNP A1 is disordered beyond Pro288-Tyr289, whereas hnRNP M extends 5 residues beyond its Pro-Tyr motif.Residues 51-64 of hnRNP M and residues 273-289 of hnRNP A1 contact a common Kapβ2 surface, with the highest overlap at their Pro-Tyr motifs (Fig. 1b). R.m.s. deviations for all Pro-Tyr atoms and for arginine guanido group atoms in the R/H/Kx (2-5) PY motifs are 0.9Å and 1.2Å, respectively, upon Kapβ2 superposition. At the N-terminal motifs, hnRNP M residues 51-54 in the basic 50-KEKNIKR-56 motif and hnRNP A1 residues 274-277 in the hydrophobic motif also overlap (main chain r.m.s. deviation ...