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. Author manuscript; available in PMC: 2017 Oct 4.
Published in final edited form as: Structure. 2016 Sep 8;24(10):1802–1809. doi: 10.1016/j.str.2016.07.018

Karyopherin-β2 recognition of a PY-NLS variant that lacks the proline-tyrosine motif

Michael Soniat 1, Yuh Min Chook 1,*
PMCID: PMC5053885  NIHMSID: NIHMS811536  PMID: 27618664

Summary

Karyopherin-β2 or Transportin-1 binds proline-tyrosine nuclear localization signals (PY-NLSs) in its cargos. PY-NLSs are described by structural disorder, overall positive charge, and binding epitopes composed of an N-terminal hydrophobic or basic motif and a C-terminal R-X2-5P-Y motif. The N-terminal tail of histone H3 binds Kapβ2 with high affinity but does not contain a recognizable PY-NLS. Crystal structure of Kapβ2-H3 tail shows residues 11–27 of H3 binding to the PY-NLS site of Kapβ2. H3 residues 11TGGKAPRK18 bind the site for PY-NLS Epitope 1 (N-terminal hydrophobic/basic motif) and is most important for Kapβ2-binding. H3 residue Arg26 occupies the PY-NLS Epitope 2 position (usually arginine of R-X2-5P-Y) but PY-NLS Epitope 3 (proline-tyrosine motif) is missing in the H3 tail. Histone H3 thus provides an example of a PY-NLS variant with no proline-tyrosine or homologous proline-hydrophobic motif. The H3 tail uses a very strong Epitope 1 to compensate loss of the often-conserved proline-tyrosine epitope.

Keywords: Karyopherin, Importin, Nuclear Import, Nuclear Localization Signal, NLS, PY-NLS, Histones

eTOC

The N-terminal tail of histone H3 binds Karyopherin-β2 (Kapβ2) with high affinity even though it lacks a recognizable proline-tyrosine nuclear localization signal (PY-NLS). Soniat and Chook determine the crystal structure of Kapβ2-H3 tail complex, showing the first example of a PY-NLS variant with no proline-tyrosine or homologous proline-hydrophobic residue motif.

Introduction

Karyopherinβs (Kaps; Importins and Exportins) are responsible for the majority of nuclear-cytoplasmic transport in eukaryotic cells (Chook and Blobel, 2001; Cook et al., 2007; Gorlich and Kutay, 1999; Weis, 2003). Each of the 20 human Kaps transports a different set of cargos by binding a nuclear localization signal (NLS) or nuclear export signal (NES) within the cargo proteins (Chook and Suel, 2011; Fung and Chook, 2014; Soniat and Chook, 2015; Xu et al., 2010). Karyopherinβ2 (Kapβ2 or Transportin-1) is an Importin that transports many protein cargos including numerous RNA binding proteins into the nucleus (Chook and Suel, 2011; Soniat and Chook, 2015; Twyffels et al., 2014; Xu et al., 2010). Kapβ2 recognizes an NLS termed the PY-NLS, which can be 15–100 residue long, very diverse in sequence and thus cannot be sufficiently described by a traditional consensus sequence. PY-NLSs are instead described by a collection of physical criteria including 1) structural disorder, 2) overall positive charge, and 3) weakly conserved sequence motifs composed of a loose N-terminal hydrophobic or basic motif and a C-terminal R-X2-5P-Y motif (Lee et al., 2006).

Sequence motifs of the PY-NLS contain three energetically important epitopes that bind Kapβ2. The N-terminal hydrophobic/basic motif is Epitope 1, the arginine of the R-X2-5P-Y motif is Epitope 2, and the proline-tyrosine (P-Y) or homologous P-Φ (Φ is hydrophobic residue) motif is Epitope 3. These binding epitopes appear structurally independent as they are connected by flexible and sequence-diverse linkers, and they are considered energetically quasi-independent as there is little energetic cooperativity between them when binding Kapβ2 (Suel et al., 2008). Very importantly, Epitopes 1, 2 and 3 contribute differently to overall binding energy in different PY-NLS peptides (Suel et al., 2008).

Most known Kapβ2 cargos contain PY-NLSs but a few cargos do not seem to have recognizable PY-NLSs (Chook and Suel, 2011; Soniat and Chook, 2015; Twyffels et al., 2014). Non-PY-NLS containing proteins that bind Kapβ2 include the core histones (H2A, H2B, H3, and H4), ribosomal proteins (rpL23A, rpS7, rpL5, rpL7), RNA-editing enzyme ADAR1, transcription factor FOXO4, and viral proteins (HIV-1 REV, HPV E6). Several groups have shown that N-terminal tails of H3 and H4 are important for nuclear import and bind Kapβ2 but their NLSs have not been characterized biochemically or structurally (Blackwell et al., 2007; Mosammaparast et al., 2002). It is currently not known how non-PY-NLS cargos bind Kapβ2. It is unclear if they carry a different NLS that binds Kapβ2 at a site distinct from the PY-NLS binding site or if they bind to PY-NLS site and are unrecognized variants of the PY-NLS.

We performed structural and biochemical analysis of Kapβ2 bound to the histone H3 tail to identify Kapβ2 binding elements in the H3 tail. Although the H3 tail does not contain a P-Y or P-ψ motif that is characteristic of PY-NLSs, our structure shows that the H3 tail is a PY-NLS variant that occupies the PY-NLS binding site of Kapβ2 with similar high affinity and contacts similar Kapβ2 residues as other characterized PY-NLSs. The H3 tail contains and uses only two of the usual three PY-NLS epitopes to bind Kapβ2: a hydrophobic-basic N-terminal segment that binds like a PY-NLS Epitope 1 and an arginine residue that is equivalent to Epitope 2 (usually arginine of the C-terminal R-X2-3-P-Y motif). The H3 tail does not have a P-Y or homologous P-ψ motif, and the binding site on Kapβ2 for the P-Y motif remains unoccupied. The structure reveals the first PY-less variant of the PY-NLS.

Results

Structure of the Kapβ2-Histone H3 Tail complex

We solved the 3.05 Å resolution structure of the H3 tail-Kapβ2 complex by molecular replacement. Crystallographic data and refinement statistics are shown in Table 1 and the overall structure of the complex is shown in Figure 1B. H3 tail-bound Kapβ2 is very similar to other PY-NLS-bound Kapβ2 proteins. Alignment of Kapβ2 residues 200–800 with Kapβ2 bound to FUSPY-NLS (4FDD), hnRNP MPY-NLS (2OT8), Nab2PY-NLS (4JLQ), hnRNP DPY-NLS (2Z5N), JKTBPPY-NLS (2Z5O), TAPPY-NLS (2Z5K) and hnRNP A1PY-NLS (2H4M) gave Cα rmsds of 1.25 –1.74 Å (Cansizoglu and Chook, 2007; Cansizoglu et al., 2007; Imasaki et al., 2007; Lee et al., 2006; Niu et al., 2012; Soniat et al., 2013; Zhang and Chook, 2012). The superhelical Kapβ2, which is composed of 20 HEAT repeats, uses its PY-NLS binding site on the concave surface of the C-terminal half of the Importin to bind residues 11–27 of the H3 tail (Figure 1B).

Table 1.

Crystallographic statistics for Kapβ2-Histone H3 tail complex.

Space group C2221
Cell dimensions:
 a, b, c (Å) 150.3, 154.2, 192.6
 α, β, γ (°) 90, 90, 90
Data Collection
 Wavelength(Å) 0.9795
 Resolution (Å) 50.00-3.05 (3.10-3.05) a
 Completeness (%) 99.9 (100.0) a
 Redudancy 7.9 (7.5) a
 Rmerge(%) 12.3 (100.0) a
 Rpim(%) 4.9 (50.9) a
 I/σI 13.2 (1.7) a
Refinement
 Resolution (Å) 47.65-3.05 (3.16-3.05) a
 No of Reflections 43618 (2172) a
 Rwork 20.6 (30.9) a
 Rfree (%) 24.9 (37.0) a
 RMS deviations
  Bond lengths (Å) 0.005
  Bond angles (°) 0.935
 Average overall B-factor (Å2) 53.3
 Solvent Content (%) 55.9
 Ramachandran plot:
  Favored region (%) 94.1
  Allowed region (%) 5.2
  Outliers (%) 0.7
Model Contents
 Protomers in ASU
  Kapβ2 2
 No. of Kapβ2 residues 1651
 No. of Kapβ2 atoms 13109
  Histone H3 tail 2
 No. of Histone H3 tail residues 34
 No. of Histone H3 tail atoms 248
 No. of water atoms 0
PDB accession code 5J3V
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a

Values in parenthesis correspond to the highest-resolution shell

Fig. 1. Sequence of the histone H3 tail and structure of the Kapβ2-H3 tail complex.

Fig. 1

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A) Domain organization of human histone H3. The segment of H3 (residues 1–47) used in co-crystalization with Kapβ2 is underlined, and the segment modeled in the crystal structure is colored cyan. B) The 3.05 Å resolution crystal structure of Kapβ2 (pink) bound to histone H3 tail (cyan). C) The Kapβ2-H3 tail interface with contacts ≤ 4.0 Å shown as dashed lines. D) The mFo-DFc difference map (contoured at 2σ, grey mesh) at the H3 tail binding site of Kapβ2. The difference map, calculated prior to modeling of the H3 tail, is shown here with the H3 tail.

Residues 11–19 of the H3 tail adopt an extended conformation while residues 20–27 form a 2-turn α-helix (Figure 1B–D). The H3 tail traces a path on Kapβ2 similar to all nine structurally characterized PY-NLSs (Cansizoglu and Chook, 2007; Cansizoglu et al., 2007; Imasaki et al., 2007; Lee et al., 2006; Niu et al., 2012; Soniat et al., 2013; Zhang and Chook, 2012). Figures 2A–D show comparison of the H3 tail to PY-NLSs of hnRNP M, hnRNP A1 (also known as the M9 sequence) and FUS (Cansizoglu et al., 2007; Lee et al., 2006). Superposition of the Kapβ2s onto the Kapβ2-H3 tail structure shows that H3 residues 11TGGKAPRK18 occupy the position of the PY-NLS epitope 1, which is usually the N-terminal hydrophobic or basic motif of a PY-NLS. The basic segment 50KEKNIKR56 is Epitope 1 of the hnRNP MPY-NLS (Figure 2A, B), the hydrophobic motifs 273FGPM276 and 508PGKM511 are Epitope 1 of the hnRNP A1PY-NLS and the FUSPY-NLS, respectively (Figures 2A, C and D). The H3 α-helix 20LATKAARK27 contains the Arg26 side chain, which occupies the position of the PY-NLS Epitope 2 arginine (conserved arginine of the R-X2-5P-Y motif; Arg60 of hnRNP M, Figure 2B; Arg284 of hnRNP A1, Figure 2C; Arg522 of FUS, Figure 2D) that forms salt bridges with acidic residues in Kapβ2. A striking difference between the H3 tail and conventional PY-NLSs is the former does not contain a PY-NLS Epitope 3, which is usually a P-Y, a homologous P-ψ or a P-X (X, any amino acid) motif (Figures 2A–D). In fact, no part of the H3 tail occupies the P-Y binding site on Kapβ2, which remains empty (Figures 2B–D).

Fig. 2. Comparison of the H3 tail with hnRNP A1PY-NLS, hnRNP MPY-NLS and FUSPY-NLS.

Fig. 2

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A) Alignment of Histone H3 tail residues 11–26 with the PY-NLSs of hnRNP M, hnRNP A1, and FUS. The sequences are aligned based on superposition of the Kapβ2-H3 tail, Kapβ2-hnRNP MPY-NLS (2OT8), Kapβ2-hnRNP A1PY-NLS (2H4M) and Kapβ2-FUSPY-NLS (4FDD) structures. Sequences in Epitope 1 are in yellow (hydrophobic motifs) or blue (basic motifs, sequences in Epitope 2 are red, and sequences in Epitope 3 are green. B) Comparison of H3 tail (cyan) with hnRNP MPY-NLS (yellow). Superposition of Kapβ2 residues 200–800 gives a Cα rmsd of 1.25 Å. PY-NLS Epitopes 1, 2 and 3 of hnRNP MPY-NLS are labeled. C) Comparison of H3 tail (cyan) with hnRNP A1PY-NLS (purple). Kapβ2s of the two complexes were superimposed (Cα rmsd 1.5 Å, superposition of Kapβ2 residues 200–800). D) Comparison of H3 tail (cyan) with FUSPY-NLS (orange). Superposition of Kapβ2 residues 200–800 - Cα rmsd of 1.74 Å.

Interactions between the H3 Tail and Kapβ2 – Epitope 1

The H3 tail contacts Kapβ2 through electrostatic, polar and hydrophobic interactions (Supplementary Table 1). Like Other PY-NLS-Kapβ2 interactions, the majority of contacts between the H3 tail and Kapβ2 involve H3 side chains rather than main chains, and all contacts occur within the PY-NLS epitopes. Figures 3A-L show interactions between four NLS peptides (H3 tail, hnRNP MPY-NLS hnRNP A1PY-NLS and FUSPY-NLS) with Kapβ2. The top row of Figure 3 (Figures 3A–D) compares interactions within Epitope 1. In the N-terminal extended portion of the H3 tail that spans residues 11–19, both Gly13 and Lys14 make hydrophobic interactions with Kapβ2 residue Trp730 (Figure 3A and Supplementary Table 1). Similar interactions to Kapβ2 Trp730 are made by 274GP275 of hnRNP A1PY-NLS and by 509GK510 of FUSPY-NLS (Figures 3C–D). The H3 13GK14 residues are followed by a string of basic residues that make many charged and polar contacts with Kapβ2. The H3 Lys14 side chain makes salt bridges with Kapβ2 residues Glu653 and Asp693, and Arg17 of H3 makes salt bridge and polar interactions with Kapβ2 Asp646, Ser591 and Ser592 (Figure 3A). Toward the end of the extended part of the H3 tail, Lys18 forms salt bridges with Kapβ2 residue Asp639 (Figure 3A). Arg17 and Lys 18 of H3 also make long-range electrostatic interactions with several acidic residues of Kapβ2 including Asp550, Glu588, Asp646, Asp653, Glu682 and Asp693 (Supplementary Table 1). The basic stretches of H3 14KAPRK18 and hnRNP MPY-NLS 52KNIKR56 make charged and polar interactions with the same set of Kapβ2 side chains (Glu653, Asp693, Asp550, Asp646, Glu588 and Glu639; Figures 3A–B).

Fig. 3. Comparison of Kapβ2-PY-NLS contacts.

Fig. 3

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Contacts (≤ 4Å) between Kapβ2 (pink) and four different PY-NLSs (the H3 tail (cyan), hnRNP MPY-NLS (yellow), hnRNP A1PY-NLS (purple) and FUSPY-NLS (orange)) are shown as dashed lines. A)–D) show Kapβ2-NLS contacts in the Epitope 1 site, E)–H) show contacts in the Epitope 2 site, and I)–L) show contacts in the Epitope 3 site.

Epitope 1 of the H3 tail (11TGGKAPRK18) is large, much like the basic Epitope 1 of hnRNP MPY-NLS, which spans 50KEKNIKR56 (Figures 2A, 3A–B). By comparison, the hydrophobic motifs in hnRNP A1PY-NLS (FGPM) and in FUSPY-NLS (PGKM) are compact (Figures 2A, 3C–D). Interestingly, based on the analysis above, Epitope 1 of the H3 tail contains elements of a hydrophobic motif, with hydrophobic contacts from residues 13GK14 to the Kapβ2 Trp730, as well as elements of a basic motif with salt bridges and hydrogen bonds from residues 14KAPRK18 to numerous acidic Kapβ2 side chains.

Interactions between the H3 Tail and Kapβ2 – Epitope 2

Following the extended portion of H3, residues 20LATKAARK27 of H3 form an α-helix that turns away from the Kapβ2 surface. H3 residues Leu20 and Thr22 make hydrophobic interactions with Kapβ2 (Supplementary Table 1). H3 Lys23 side chain makes long-range electrostatic interactions with Kapβ2 residues Glu509 and Asp550. At the C-terminal end of the H3 helix, Arg26 and Lys27 form salt bridges with Kapβ2 residues Glu509 and Asp543. The H3 tail helix is oriented ~35° away from the C-terminal portions of the hnRNP M, hnRNP A1 and FUS PY-NLSs (Figures 2B–D). However, despite the different direction that the H3 tail helix takes, its Arg26 side chain reaches back to make salt bridges and polar contacts with the same Kapβ2 residues (Glu509, Asp643 and Thr547) as the conserved Epitope 2 arginines of the other PY-NLSs (Arg60 of hnRNP MPY-NLS, Arg284 of hnRNP A1PY-NLS and Arg522 of FUSPY-NLS; Figures 2B–D, 3E–H).

The PY-NLS of the protein Fused in Sarcoma (FUS) also contains a small helix (Niu et al., 2012; Zhang and Chook, 2012). Like the H3 helix, several side chains (including the Epitope 2 arginine) on one face of the FUS PY-NLS helix interact with Kapβ2 (Figure 2D, 3E, 3H). We had previously proposed that the entire FUS helix is a single structural unit that makes up Epitope 2 of that PY-NLS (Niu et al., 2012; Zhang and Chook, 2012)). Similarly, we propose here that the H3 helix and not just Arg26 is Epitope 2. However, unlike the H3 helix, which turns away from the Kapβ2 surface, the FUS helix follows the paths of typical PY-NLSs close to the Kapβ2 surface (Figures 2D, 3E, 3H).

The NLS in the H3 tail that binds Kapβ2 resembles PY-NLSs structurally and binds Kapβ2 like a PY-NLS even though it does not contain the canonical P-Y or P-α dipeptide motif (Figure 2A). The H3 NLS for Kapβ2 does not use any binding element in place of the prevalent and often conserved P-Y epitope (Figure 2B–D, 3I).

Mutagenic analysis of the Kapβ2-Histone H3 tail interactions

The H3 tail binds Kapβ2 with high affinity (KD of 77.1 nM, measured by isothermal titration calorimetry (ITC)) comparable with known Kapβ2–PY-NLS interactions (Cansizoglu et al., 2007; Lee et al., 2006; Suel and Chook, 2009; Suel et al., 2008; Zhang and Chook, 2012) (Table 2 and Supplementary Figures 1 and 2). We systematically mutated H3 residues that contact Kapβ2 to examine the distribution of binding energy in the peptide. In the extended portion of H3, only basic side chains (Lys14, Arg17 and Lys18) contact Kapβ2. Similarly, many interactions between the H3 helix involve its basic side chains. Therefore, we mutated all basic residues in the extended part of H3 (mutant H3 tail(K14A,R17A,K18A)), all basic residues in the helix (mutant H3 tail(K23A,R26A,K27A)) and all basic residues in the H3 NLS (mutant H3 tail(K14/R17/K18/K23/R26/K27 to As)), and measured affinities for Kapβ2 by ITC. The H3 tail(K14A,R17A,K18A) mutant no longer binds Kapβ2 but mutating basic residues in the H3 helix (H3 tail(K23A,R26A,K27A)) decreased affinity by only ~1.5 fold (Table 2 and Figure 2). Mutation of all basic residues (mutant H3 tail(K14/R17/K18/K23/R26/K27 to As)) resulted in no Kapβ2 binding (Table 2 and Figure 2).

Table 2.

Binding affinities of Kapβ2 with H3 tails by ITC.

Ligand KD (nM)
MBP-H3 Tail(1–47):
wild-type 77.1±15.5
K14A, R17A, K18A ND
K14/R17/K18/K23/R26/K27 to As ND
K23A, R26A, K27A 119.4±39.7
K14A ND
R17A 388.9±12.3
K18A 119.6±12.6
K23A 147.5±16.3
R26A 122.5±16.1
K27A 132.7±11.2
MBP-H3 Tail(1–28) 104.5±11.8
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We also mutated individual basic residues within the H3 segment that binds Kapβ2. Lys14 of H3 contributes the most to Kapβ2 binding as the H3 tail(K14A) mutant shows no Kapβ2 binding (Table 2 and Figure 2). The H3 tail(R17A) mutant binds Kapβ2 ~5-fold weaker while individual mutations of K18A, K23A, K26A and K27A each shows less than 2-fold decrease in affinity (Table 2 and Figure 2). Truncation of the H3 tail to residues 1–28 had less than a negligible 1.5-fold effect on Kapβ2 binding (KD of 77 nM for H3 residues 1–47 vs. KD of 105 nM for H3 residues 1–28), consistent with structural observations that binding elements for Kapβ2 resides within residues 11–27 of the H3 tail (Table 2). In summary, the N-terminal extended segment, which makes up Epitope 1 of the H3 tail (11TGGKAPRK18) is important for binding Kapβ2. Within Epitope 1, the H3 residue Lys14 appears to be the binding hotspot.

Discussion

The H3 tail has no discernible PY-NLS but binds the PY-NLS site of Kapβ2 by contacting many of the same Kapβ2 residues as typical PY-NLSs. PY-NLSs usually use three modular epitopes to bind Kapβ2 (Cansizoglu et al., 2007; Lee et al., 2006; Suel et al., 2008; Xu et al., 2010). Combinatorial mixing of energetically weak and strong motifs PY-NLS Epitopes 1, 2 and 3 results in a range of Kapβ2 affinities suitable for nuclear import and generates large sequence diversity of PY-NLSs (Suel et al., 2008). The H3 tail is an example of a novel combinatorial use of PY-NLS epitopes. The very strong hydrophobic-basic Epitope 1 in H3 and a possibly moderate-strength helical Epitope 2 appear to have obviated the need for the canonical C-terminal P-Y Epitope 3. The very strong Epitope 1 also contains two acetylation sites as 20–30% of cytoplasmic H3 is acetylated at Lys14 (Kapβ2-binding hotspot) and/or Lys18 (Jasencakova et al., 2010). Lys14 acetylation abolished Kapβ2 binding, suggesting that the modification may attenuate nuclear import of the small pool of cytoplasmic H3/H4 acetylated at H3 Lys14 (Soniat et al., 2016).

There are other Kapβ2 cargos with no recognizable PY-NLSs (Soniat and Chook, 2015). We envision three scenarios for Kapβ2 binding to non-PY-NLS sequences. In the first, some cargos may have entirely distinct signals that bind a different Kapβ2 site. In the second scenario, cargos may have P-Y-related motifs that bind the P-Y binding site of Kapβ2. For example, a Glu-Arg dipeptide in the NLS of human adenosine deaminase acting on RNA 1 enzyme (ADAR1) was suggested to bind as Epitope 3 to the P-Y site of Kapβ2 (Barraud et al., 2014). Our third scenario is the true PY-less PY-NLS, which does not use an Epitope 3. Epitopes 1 and 2 in H3 binds Kapβ2 with sufficient affinity to be a functional NLS. Functional PY-NLSs may also contain other 2-epitope combinations: either Epitopes 1+3 (no Epitope 2) or Epitopes 2+3 (no Epitope 1). Unusually strong individual PY-NLS Epitopes 1 2 or 3 may also be used alone as NLS.

Both the N-terminal tail of histone H4 and the ribosomal protein rpL23A also bind Kapβ2 but do not have P-Y motifs (Baake et al., 2001; Blackwell et al., 2007; Campos et al., 2010; Greiner et al., 2004; Jakel and Gorlich, 1998; Johnson-Saliba et al., 2000; Mosammaparast et al., 2002; Muhlhausser et al., 2001). Kapβ2 binds H4 tail residues 5–20 but the affinity is ~11-fold weaker than the H3 tail (H4, KD 871 nM vs. H3, KD 77 nM) (Soniat et al., 2016). rpL23A uses a 43-residue β-like import receptor binding (BIB) sequence to bind Kapβ2 and other Importins in cell lysates, but biochemical studies with recombinant proteins are not available (Jakel and Gorlich, 1998). Comparison of the H3, H4 tail segments that bind Kapβ2 and the BIB of rpL23A shows some similarity (Supplementary Figure 3). As in H3, the H4 tail may use a hydrophobic-basic Epitope 1 and a helical Epitope 2 to compensate for the loss of Epitope 3. In contrast, potential PY-NLS Epitopes 1 and 2 in the rpL23A BIB show less resemblance to those in the H3 tail. While rpL23A 59RQPKYPRK66 contains several basic residues and thus resembles a basic Epitope 1, and arginine and lysine residues may fulfill the role of the conserved arginine in Epitope 2, the C-terminal segment 67SAPRRNKL84 lacks helical propensity and may not be as strong a binder as the helical Epitope 2 of the H3 or H4 tails. It remains to be determined if rpL23A binds Kapβ2 as well as typical PY-NLSs or the weak PY-NLS variant in H4 tail.

The diversity in PY-NLS sequence, length and conformation is striking. Construction of the peptides from multiple binding epitopes that are connected by linkers allows for sequence diversity within each epitope that collectively produces very ‘loose’ PY-NLS sequences. Flexible linkers make few or no contacts with Kapβ2, and therefore neither their sequences nor lengths need to be conserved. Furthermore, any individual epitope may be jettisoned if the other epitope(s) can provide sufficient Kapβ2-binding energy. Finally, the ‘looseness’ of PY-NLSs is also evident in their different paths and conformations when bound to Kapβ2 (Figures 2B–D). Although the majority of contacts by different PY-NLSs are to the same Kapβ2 residues, they involve mostly NLS side chains. This mode of interaction removes significant constrains on the PY-NLS backbones, unlike Impα and Impβ that bind classical-NLS and IBB peptides in very consistent positions and conformations by restraining the NLS main chain.

Several Importins other than Kapβ2 can also import H3 (Baake et al., 2001; Blackwell et al., 2007; Campos et al., 2010; Greiner et al., 2004; Johnson-Saliba et al., 2000; Mosammaparast et al., 2002; Muhlhausser et al., 2001). Moreover, the primary H3 importer in cells is Importin-4 and Kapβ2 is merely one of several ‘backup’ importers. The H3 tail can target heterologous proteins to the nucleus, and simultaneous removal of H3 and H4 tails prevented nuclear localization of the histone pair (Blackwell et al., 2007; Ejlassi-Lassallette et al., 2011). Despite importance of the H3 tail, the histone fold domain of H3 may also contribute to Importin binding. A recent study shows that both histone tails and histone fold domains are important for Importin binding (Soniat et al., 2016). A complete picture of how H3 is transported awaits structures of Importins bound to full-length H3/H4 dimers.

The H3 tail binds Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9 and Impα with affinities ranging from 50–500 nM (Soniat et al., 2016). H3 residues 11–27 are also important for binding the other Importins (Soniat et al., 2016). The 17-residue segment contributes most of the binding energy for Impβ, Kapβ2 and Imp4 while interactions with Imp5, Imp7, Imp9 and Impα are more complex as they also involve a downstream C-terminal basic element (Soniat et al., 2016). The ability of histone tails to bind multiple Importins is shared by BIB sequences of ribosomal proteins, which were suggested to be ancestral nuclear import signals that existed before Importins diverged to have specialized NLS binding sites (Jakel and Gorlich, 1998). Even though Kapβ2 has evolved to bind the PY-NLS, it appears to have retained binding to the ancestral BIB and the similar histone tails that had not evolved a P-Y motif.

Experimental Procedures

Cloning, Expression, and Purification of H3 tail and Kapβ2

GST-fusion constructs were generated by inserting PCR fragments corresponding to the regions of the genes of interest into the pGEX-TEV plasmid, which is a pGEX4T3 vector (GE Healthcare, UK) modified to include a TEV cleavage site (Chook and Blobel, 1999). 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) (Cansizoglu et al., 2007; Lee et al., 2006). GST-fusion construct of the Histone H3 tail (1–47) was kindly provided by B. Li (UT Southwestern, TX). MBP-fusion constructs of the H3 tail (1–47) and shorter fragments were subcloned into the pMALTEV vector (pMAL (New England BioLabs, Ipswich, MA) with TEV cleavage site) (Chook et al., 2002). H3 tail mutations were made by site-directed mutagenesis using a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), and all constructs were sequenced.

Human Kapβ2 was expressed as a GST-fusion protein. Human H3 tail (residues 1–47) was also expressed as GST-fusion and MBP-fusion proteins. All recombinant proteins were expressed separately in BL21 (DE3) E. coli cells (induced with 0.4 mM isopropyl-α-d-1-thiogalactoside (IPTG) for 12 hours at 25 °C). Bacteria were lysed with the EmulsiFlex-C5 cell homogenizer (Avestin, Ottawa, Canada) in buffer containing 50 mM Tris pH 7.5, 200 mM NaCl, 20% glycerol, 2 mM DTT, 1mM EDTA, and protease inhibitors.

GST-Kapβ2 proteins were purified by affinity chromatography on GSH sepharose (GE Healthcare, UK), eluted with buffer containing 50 mM Tris pH 7.5, 75 mM NaCl, 20% glycerol, 2 mM DTT and 20 mM L-Glutathione. GST-Kapβ2 were cleaved with TEV protease, and Kapβ2 was further purified using ion-exchange and gel filtration chromatography in TB buffer (20 mM HEPES pH 7.3, 200 mM sodium chloride, 2 mM DTT, 2 mM magnesium acetate, 10% glycerol, 1 mM EGTA).

To purify MBP-H3 tail proteins, bacterial lysates were incubated with amylose beads (New England BioLabs, Ipswich, MA) and the fusion proteins eluted with buffer containing 20 mM Tris pH 7.5, 50 mM NaCl, 2 mM EDTA, 2 mM DTT, 10% glycerol, and 10 mM maltose. Eluted proteins were further purified by ion-exchange chromatography.

Structure determination of the Kapβ2 bound to the H3 Tail

GST-H3 tail (residues 1–47) and Kapβ2 (residues 337–367 replaced with a GGSGGSG linker) were mixed at molar ratio of 5:1. GST was removed with TEV protease and the Kapβ2-H3 tail complex further purified by gel filtration in buffer containing 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 Kapβ2-H3 tail complex was concentrated to 15 mg/mL for crystallization.

Kapβ2-H3 tail crystals were obtained by sitting drop vapor diffusion at 20°C (0.4 μL protein + 0.4 μL reservoir solution) with reservoir solution of 200 mM NaF, 20% PEG3350. Crystals were cryo-protected by addition of ~20% (v/v) ethylene glycol, and flash-cooled by immersion in liquid nitrogen. Many crystals did not yield useful diffraction, but few diffracted to 3.05-Å resolution. X-ray 0.9795 Å wavelength diffraction data was collected at the Advance Photon Source 19ID beamline in the Structural Biology Center at Argonne National Laboratory. Date was indexed, integrated, and scaled using HKL3000 (Minor et al., 2006). The structure was determined by molecular replacement using PHASER with a search model of human Kapβ2 (A chain of PDB Code 2QMR) (Cansizoglu and Chook, 2007). Several rounds of refinement using PHENIX and manual model building with Coot were performed (Adams et al., 2010; Emsley et al., 2010). Residues 11–27 of H3 tail were built into the electron density maps at the last stages of the refinement. The final model of the Kapβ2-H3 tail complex shows excellent stereochemical parameters based on Molprobity suite in PHENIX (Chen et al., 2010) (Table 3). Illustrations were prepared with PyMOL (Schrodinger).

Measuring dissociation constants with isothermal titration calorimetry

Binding affinities of MBP-H3 tail proteins to Kapβ2 were measured using isothermal titration calorimetry (ITC). ITC experiments were performed with a Malvern ITC200 calorimeter (Malvern Instruments, Worcestershire, UK). Proteins were dialyzed against buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, and 2 mM β-mercaptoethanol. 200–400 μM MBP-H3 tail proteins were titrated into a sample cell containing 20–40 μM recombinant Kapβ2. ITC experiments were performed at 20°C with 19 rounds of 4-μL injections. Data were plotted and analyzed using NITPIC and Sedphat and the data visualized using GUSSI. Averages of three ITC runs were plotted with standard errors in histograms generated by GraphPad Prism.

Supplementary Material

1
2

Highlights.

  • H3 tail does not have a recognizable PY-NLS but binds Kapβ2 with high affinity.

  • Crystal structure of Kapβ2-H3 tail shows H3 tail binding in Kapβ2 PY-NLS site.

  • H3 tail uses typical PY-NLS Epitopes 1 and 2 but is missing canonical P-Y motif.

  • Very strong Epitope 1 compensates loss of the proline-tyrosine Epitope 3.

Acknowledgments

We thank Bing Li for histone constructs, Chad Brautigam and Thomas Scheuermann from the Macromolecular Biophysics Resource at UTSW for assistance in ITC experiments, Diana Tomchick and James Chen from Structural Biology Laboratory at UTSW for assistance in crystallographic data collection, and Ho Yee Joyce Fung and Szu-Chin Fu for comments. The use of SBC 19ID beamline at Advanced Photon Source is supported by U.S. Department of Energy contract DE-AC02-06CH11357. This work is funded by NIH R01 GM069909 (Y.M.C.), NIH U01 GM98256-01 (YMC), Welch Foundation Grant I-1532 (Y.M.C.), Leukemia and Lymphoma Society Scholar Award (Y.M.C.) and the University of Texas Southwestern Endowed Scholars Program (Y.M.C.).

Footnotes

Author Contributions

M.S. conducted the experiments. M.S. and Y.M.C. designed the experiments and wrote the paper.

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Supplementary Materials

1
NIHMS811536-supplement-1.pdf (773.3KB, pdf)
2
NIHMS811536-supplement-2.pdf (775.8KB, pdf)

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