Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2
- First Online:
- Cite this article as:
- Soniat, M., Sampathkumar, P., Collett, G. et al. J Struct Funct Genomics (2013) 14: 31. doi:10.1007/s10969-013-9150-1
- 456 Views
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.
KeywordsKaryopherinImportinNuclear importNuclear localization signalNucleocytoplasmic transportNuclear poreNab2
Karyopherin β proteins (Kaps; also known as Importins and Exportins) are responsible for the majority of nucleocytoplasmic transport in eukaryotic cells. At least 20 members of the Kap family have been identified in humans, whereas 14 Kaps are known in Saccharomyces cerevisiae. Each Kap binds a unique set of cargos and targets them to the nuclear pore complex. Kaps bind nuclear localization signals (NLSs) or nuclear export signals (NESs) in cargo proteins to direct them in and out of the nucleus, respectively. Kap-cargo interactions and transport directionality are in turn regulated by the Ran GTPase nucleotide cycle .
The Karyopherin β2 (Kapβ2; also known as Transportin) importin recognizes a class of NLS in its cargos termed the PY-NLS [2–4]. These 15- to 100-residue long sequences are diverse and cannot be sufficiently described by a traditional consensus sequence. PY-NLSs are instead described by a collection of physical rules that include the requirements for intrinsic structural disorder, overall basic character, and a set of sequence motifs. PY-NLS motifs consist of an N-terminal hydrophobic or basic motif and a C-terminal RX2-5PY motif [4–6].
All previous structures of PY-NLSs bound to Kapβ2 are of signals that contain the canonical PY dipeptide motif. The PY-NLS of Nab2 is unusual in that it contains a homologous PL dipeptide motif at its C-terminus. Mutagenesis studies suggest that some PY dipeptides in PY-NLSs can be replaced by PΦ dipeptides where Φ is any hydrophobic residue without losing binding affinity for the Kap . Here we report the 3.05-Å-crystal structure of Kapβ2 bound to the PY-NLS of Nab2, which shows for the first time the homologous PL dipeptide motif. The structure explains how an aliphatic hydrophobic residue is able to substitute structurally for the conserved tyrosine in a PY-NLS.
Materials and methods
Protein expression, purification, and complex assembly
Human Kapβ2 (Uniprot ID U72069) was expressed in pGEX-TEV vector [pGEX-4T3 (GE Healthcare) with a TEV cleavage site] as a GST fusion protein and purified as previously described . Kapβ2 with a truncated loop, which does not interfere with NLS binding, was used for crystallization (residues 337–367 of Kapβ2 were replaced with a GGSGGSG linker) . Residues 205–242 of S. cerevisiae Nab2 (Nab2PY-NLS; Nab2, Uniprot ID P32505) were also expressed as a GST fusion protein . GST-Nab2PY-NLS was purified by affinity and ion exchange chromatography. GST-Nab2PY-NLS and Kapβ2 were mixed at molar ratio of 5:1. The GST tag was removed with TEV protease and the complex further purified by gel filtration in buffer composed of 20 mM HEPES, pH 7.3, 110 mM potassium acetate, 2 mM DTT, 2 mM magnesium acetate, and 1 mM EGTA with 20 % (v/v) glycerol. The complex was concentrated to 13 mg/mL for crystallization.
Crystallization and structure determination of the Kapβ2·Nab2PY-NLS complex
Crystallographic statistics for HsKapβ2·ScNab2PY-NLS complex
a, b, c (Å)
132.2, 172.4, 68.4
α, β, γ (°)
90, 90, 90
Matthew’s coefficient (Å3/Da)
Solvent content (%)
Number of unique reflections
Number of reflections in Rfree set
Overall completeness (%)
RMSD bond lengths (Å)
RMSD bond angles (°)
Molprobity  Ramachandran:
Favored region (%)
Allowed region (%)
Mean overall B-factor
Protomers in ASU
No. of HsKapβ2 residues
No. of HsKapβ2 atoms
No. of ScNAB2-NLS residues
No. of ScNAB2-NLS atoms
No. of water atoms
PDB accession code
Results and discussion
Structure of the Kapβ2·Nab2PY-NLS complex
We have determined the 3.05 Å crystal structure of human Kapβ2 bound to the PY-NLS segment of S. cerevisiae Nab2 that spans residues 205–242 (Fig. 1b). Kapβ2, as shown previously [11, 17], is a superhelical protein composed of 20 α-helical HEAT repeats. Each HEAT repeat is composed of two antiparallel α-helices, A and B, each lining the convex and concave sides of the superhelix, respectively. Seven residues of the Nab2PY-NLS (residues 234–240) are modeled (Fig. 1c) and shown to bind the previously described PY-NLS binding site in the C-terminal arch of Kapβ2 [4–6, 17]. Residues 234–240 of the Nab2PY-NLS peptide bind in extended conformation, tracing a path along the concave surface of Kapβ2 similar to other structurally characterized PY-NLSs, such as that from hnRNP A1 (Fig. 1d, e) . Residues 204–233 and 241–242 of the bound Nab2PY-NLS peptide were not modeled due to weak electron density.
Further N-terminus, Phe-236 of Nab2 also makes hydrophobic interactions with Ala-499, Glu-498 and Trp-460 sidechains of Kapβ2. The Phe236 sidechain is also positioned close to Pro-238 (within 4.5 Å). Similar conformations were observed for the equivalent position in PY-NLSs of hnRNP D, hnRNP M and TAP/NXF1 where a phenylalanine is also present (Fig. 2b) [4, 5, 17]. Previous biochemical studies have shown that Phe-236 contributes significantly to Kap104-Nab2 interactions and that a hydrophobic residue at this position is important . Phe-236 likely contributes entropically through intramolecular interactions that preorganize the PL motif for Kapβ2 binding. The neighboring Arg-235, which is the N-terminal arginine of the RX2-5PY/L motif, makes multiple salt bridges and hydrogen bonds with Asp-543, Thr-506, Glu-509, and Thr-547 of Kapβ2 (Fig. 2a). Beyond residue Thr-234, electron density is too weak to model the N-terminal portion of the Nab2 PY-NLS.
Protein data bank codes
Atomic coordinates and structure factors of the Kapβ2·Nab2PY-NLS complex were deposited to the PDB on 12 March 2013 with accession codes 4JLQ. The NYSGRC target identifiers for the PSI:Biology targets Kapβ2 and Nab2PY-NLS are “NYSGRC-020458” and “NYSGRC-020441”, respectively.
We thank Z. Zhang for advice on protein purification and M. Rout for discussions. This work is funded by the National Institutes of Health U01 GM98256-01 (YMC, ASC), U54 GM094662 (SCA) and R01-GM069909 (YMC), Welch Foundation (I-1532; YMC), Leukemia and Lymphoma Society Scholar award (YMC), CPRIT (RP120352; YMC) and UT Southwestern Endowed Scholars Program (YMC). This publication was made possible by the Center for Synchrotron Biosciences grant, P30-EB-009998, from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Basic Energy Sciences. Access to the LRL-CAT beam line facilities at Sector 31 of the APS was provided by Eli Lilly, which operates the facility.