Crystal structure of the NEP C-terminal domain

Although NEP^1–54 failed to crystallize and NEP^54–121 yielded crystals of only poor quality, we could obtain well-diffracting crystals by digesting a slightly shorter construct (NEP^54–116) with subtilisin to obtain the C-terminal fragment NEP^59–116 (Figure 1B). Crystals belong to space group P3₂21, with cell parameters a = b = 82.7 Å, c = 61.1 Å, and two molecules per asymmetric unit. Phases obtained using two heavy-atom derivatives resulted in a 2.6 Å experimental map which was readily interpreted (Table I). The final model, with R_cryst = 0.176 and R_free = 0.244, includes residues 63–116 and 48 water molecules. Residues 59–62 are disordered.

Table I.

Data collection, phasing and refinement statistics

	Native	PtCl4 derivative (I)	PtCl4 derivative (II)	OsCl6 derivative
Data collection
Resolution (Å)	2.6 (2.74–2.60)	3.50 (3.69–3.50)	3.50 (3.69–3.50)	2.8 (2.95–2.80)
Completeness	0.996 (1.000)	0.991 (0.986)	1.000 (1.000)	0.965 (0.817)
Multiplicity	3.8 (3.8)	6.7 (6.8)	14.9 (15.5)	6.0 (3.1)
I/σ	6.0 (2.0)	5.5 (2.3)	3.4 (2.0)	4.9 (3.9)
Rmerge	0.090 (0.32)	0.083 (0.326)	0.102 (0.330)	0.093 (0.168)
Ranomalous		0.065 (0.148)	0.063 (0.107)	0.043 (0.147)
Phasing
Overall FOM	Overall 0.33	At 4.6 Å: 0.71	At 3.4 Å: 0.28	At 2.9 Å: 0.06
FOM after RESOLVE	Overall 0.54	At 4.6 Å: 0.89	At 3.3 Å: 0.60	At 2.6 Å: 0.15
Refinement
No. of reflections working set	6962
Rcryst	0.176 (0.225)
Rfree	0.244 (0.319)
Average B factor (Å2)	43.5
R.m.s. bonds (Å)	0.043
R.m.s. angles (°)	3.3

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A test set containing 5% of the reflections was used throughout the refinement. Values for the highest resolution bin are given in parentheses.

The C-terminal NEP domain forms a helical hairpin with approximate dimensions 40 × 25 × 15 Å (Figure 2A). Residues 64–85 and 94–115 comprise helices C1 and C2, respectively, while residues 86–93 form the interhelical turn. The two helices are equal in length and nearly perfectly antiparallel, giving the structure a remarkably flat appearance. Because of their shallow crossing angle (14°), the two helices share extensive contacts over their entire length, the majority of which are hydrophobic in nature. These are mediated by a total of 12 hydrophobic and four polar residues, with the two helices contributing one or two side chains from each of their six helical turns (Figure 2A and B). The hairpin is further stabilized by interactions involving three arginine residues from helix C1: Arg84 forms a hydrogen bond with glutamine Gln96, Arg77 forms a bifurcated salt bridge with glutamates Glu74 and Glu110, and Arg66 has its guanidium group favourably located near backbone carbonyl groups at the C-terminus of helix C2, such that its positive charge interacts with the helix dipole (Figure 2A). The eight residues forming the interhelical turn are well ordered (average B factors resemble those of helical residues) and include two hydrophobic residues partially buried between the helices. Thus the C-terminal domain is a highly compact unit with little flexibility, explaining its resistance to proteolytic digestion.

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Fig. 2. Structure of the NEP C-terminal domain. (A) Ribbon diagram. The six layers of hydrophobic residues involved in interhelical contacts are shown. Also shown are the hydrogen bond between Arg84 and Gln96, the capping interaction between Arg66 and the C-terminus of helix C2, and the salt bridge involving Arg77, Glu74 and Glu110. For clarity, not all interhelical contacts are shown. (B) Sequence and secondary structure of NEP. Residues conserved between influenza A and B viruses are highlighted in yellow and grey. Residues predicted by the program PHD to be helical with >90% probability are labelled H. The NES motif and residue Trp78 implicated in M1 binding are boxed. Arrows mark limits of the proteolytically resistant fragment; residues in italics are absent from the construct used for structure determination; disordered residues are indicated by the dotted line. Contacts between helices C1 and C2 within one monomer, and those between two monomers of the dimer are indicated as follows: green squares, van der Waals contact involving a hydrophobic residue (filled square) or the aliphatic moiety of a polar residue (empty square); red square, salt bridge; filled black square, hydrogen bond; empty black square, capping interaction of Arg66 with C-terminus of helix C2. Typically, residues from helix C1 interact with those from helix C2 of the same or an adjacent layer. (C) Schematic diagram showing side-chain distribution. Helix C1 is viewed from C- to N-terminus and helix C2 from N- to C-terminus. Hydrophobic, polar, acidic and basic residues are coloured green, black, red and blue, respectively. The side chains of Arg66 and Arg84 emerge from the polar/charged face; those of Gln71, Trp78 and Arg86 point as shown by the arrows. (D) Surface properties of the C-terminal domain. Upper panels: electrostatic surface potential plotted from –10 kT (red) to +10 kT (blue). Lower panels: distribution of hydrophobic (green) and hydrophilic (yellow) side chains. The upper and lower left panels are in the same orientation as (A). The figure was produced using GRASP (Nicholls et al., 1991).

The helical hairpin is strikingly amphipathic, with one face hydrophobic and the other hydrophilic (Figure 2C and D). The former contains 16 of the 21 hydrophobic residues present in the domain; these form a broad swathe that extends from the top to the bottom of the hairpin (Figure 2D, bottom left). In contrast, the opposite face bears 15 of the domain’s 19 charged residues, almost all of which are located on helix C1. Six glutamate side chains cluster near the centre of this face, whereas basic residues localize to the top and bottom (Figure 2D, top right). This face also contains five hydrophobic residues (Figure 2D, bottom right). Four of these (Ile97, Met100, Leu103 and Leu107) form a continuous groove on the surface; the fifth is a prominently exposed tryptophan (Trp78) located in the centre of the glutamate cluster.

The isolated NEP C-terminal domain is a dimer

In the crystal structure, the hydrophobic face of one helical hairpin is buried against that of a second hairpin, related to the first by a non-crystallographic dyad. The two form an antiparallel four-helix bundle, with helix C1 from one hairpin packing against helix C2 of the other, and all four N- and C- termini located at one end (Figure 3A). The interface between the two monomers buries an accessible surface area of 1268 Å², representing 27% of the total surface area. Comparable values have been observed for homodimers of a similar molecular weight (Jones and Thornton, 1996) and for protein–protein interfaces in general (Lo Conte et al., 1999). Twenty-four residues from each hairpin interact with residues from the other monomer (Figure 2B). Most of these residues mediate hydrophobic contacts, and with only a few exceptions are identical to those mediating intramonomer contacts between the C1 and C2 helices. These residues are located near the 2-fold axis in each of the six layers defined by the turns of the four helices (Figure 3A). Interactions within and between layers give the helical bundle a tightly packed hydrophobic core, similar to that observed in the interior of globular proteins. Additional interactions stabilizing the dimer are a hydrogen bond between Glu95 and the backbone carbonyl group of Val83, and a salt bridge between Lys72 and glutamates Glu108 and Glu112 (Figure 3A).

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Fig. 3. The NEP C-terminal domain dimer. (A) Ribbon diagram of the four-helix bundle. The two monomers belong to the same asymmetric unit, related by a non-crystallographic dyad running nearly parallel to the individual helices. Six successive layers of interacting side chains define the hydrophobic core. Each of the helices in the bundle contributes one or more side chains per layer, with symmetry-related residues in the same layer. Not all residues in the dimerization interface are shown (but see Figure 2B). Hydrophobic residues in the dimer interface are identified in the legend to the right. Also shown are residue Trp78 implicated in M1 binding, the hydrogen bond between Glu95 and the backbone carbonyl of Val83, and the salt bridge involving Lys72, Glu108 and Glu112. (B) Analytical gel filtration of NEP and NEP^54–121. Arrows (from left to right) indicate elution volumes of the globular reference proteins bovine serum albumin, ovalbumin, chymotrypsinogen A and RNase A. The elution volumes of NEP (14.5 kDa) and NEP^54–121 (8.5 kDa) are nearly identical, suggesting that the latter is a dimer in solution. (C) Cross-linking experiment. NEP or NEP^59–116 was incubated with the indicated amount of EGS for 1 h on ice. Reactions were quenched by adding 100 mM Tris and analyzed by denaturing gel electrophoresis. Squares indicate the positions of monomers and dimers. Small amounts of the dimeric NEP and tetrameric NEP^59–116 species at high EGS concentrations are probably due to transient interactions between molecules in solution.

The many interactions between the two monomers and the hydrophobic nature of the interface suggest that the observed dimer is not merely an artefact of crystallization, but represents the state of the NEP C-terminal fragment in solution. Indeed, unlike the monomer, the dimer has relatively few solvent-exposed hydrophobic residues and thus is more consistent with the highly soluble nature of this fragment. To verify this, we performed analytical gel filtration on NEP^54–116 and on full-length NEP, which is known to be monomeric (Lommer and Luo, 2002). Despite their nearly 2-fold difference in molecular weight (8.5 versus 14.5 kDa), both NEP^54–116 and full-length NEP elute at the same volume, corresponding to that expected for a globular protein of 15 kDa (Figure 3B). Given that the crystal structures of monomeric and dimeric NEP^59–116 are both highly compact (i.e. essentially globular), the result is consistent with the C-terminal fragment being dimeric in solution. This conclusion is further supported by the results of a cross-linking study. NEP treated with the cross-linking reagent ethylene glycol bis-succinimidylsuccinate (EGS) yields essentially a single band on a denaturing gel, consistent with a monomeric species (Figure 3C, lanes 1–4). In contrast, treatment of NEP^59–116 results in the appearance of a second band whose size corresponds to that expected for a dimer (lanes 5–8).

In these experiments, identical results were obtained regardless of whether the C-terminal fragment was expressed from a plasmid or obtained by proteolysis of the full-length protein. Therefore we conclude that proteolytic degradation of the N-terminal domain leads to spontaneous dimerization of the C-terminal domain. A likely explanation is that the N-terminal domain packs against the hydrophobic face of the C-terminal domain in the structure of full-length NEP, sterically hindering access to the dimerization interface; thus proteolysis would remove this steric hindrance. Burial of the hairpin’s hydrophobic face by the N-terminal domain would also explain why full-length NEP is highly soluble in the absence of detergents.

NEP/M1 binding is mediated by the NLS motif of M1 and residue Trp78 of NEP

In order to localize the sites of interaction between NEP and M1, we set out to find mutants deficient for binding activity. M1 is known to consist of two domains: an N-terminal domain (residues 1–164), for which the crystal structure is known (Sha and Luo, 1997; Arzt et al., 2001), and a C-terminal domain (residues 165–252) that mediates vRNP binding (Baudin et al., 2001). Pulldown assays performed as above show that NEP^54–116 associates with the N-terminal, but not the C-terminal, domain of M1 (Figure 4B; data not shown). Because NEP has previously been reported to bind an M1 fragment lacking residues 1–88 (Yasuda et al., 1993), we conclude that a critical NEP binding epitope is located within the M1 N-terminal domain between residues 89 and 164. In the crystal structure of M1^1–164, residues 89–164 form a four-helix bundle constituting a compact subdomain. This subdomain includes an exposed basic motif ¹⁰¹RKLKR¹⁰⁵ that mediates binding to negatively charged liposomes (Baudin et al., 2001) and is recognized by the cellular import machinery as a nuclear localization signal (NLS) (Ye et al., 1995; Liu and Ye, 2002). We tested the ability of NEP to bind to M1^mutNLS, an M1 mutant in which the four basic residues of the NLS are replaced by alanines (¹⁰¹AALAA¹⁰⁵). This mutant was found to have severely reduced NEP binding affinity (Figure 4C). Failure to bind NEP cannot be ascribed to misfolding of the mutant protein, as its N-terminal domain (residues 1–164) has essentially the same crystal structure as that of wild-type M1 (unpublished results). It has been reported that M1 protein bearing a single mutation of either Lys102 or Lys104 is no longer imported into the nucleus of infected cells and results in lethality 72 h after transfection, whereas mutation of R101 or R105 has little effect on M1 nuclear import or RNP export (Liu and Ye, 2002). To assess the relative contribution of the two half-sites of the NLS motif to NEP binding, we constructed double point mutants of M1 in which either residues Arg101 and Lys102 or residues Lys104 and Arg105 were simultaneously replaced by alanine residues (mutants M1^{R101A, K102A} and M1^{K104A, R105A}, respectively). In pulldown experiments, both mutants bound NEP more weakly than wild-type M1 but more strongly than the M1^mutNLS mutant which lacks all four basic residues (compare Figure 4A and C with D and E). This indicates that at least one basic residue from each NLS half-site contributes to NEP binding, and that residues in both half-sites are required for full binding activity. Masking of the NLS motif by NEP may ensure that newly exported progeny vRNPs destined for virus assembly at the plasma membrane are not reimported into the nucleus.

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Fig. 4. NEP residue Trp78 and the NLS of M1 mediate NEP–M1 binding. Nickel–agarose beads preincubated with a His-tagged NEP construct (‘bait’) (even-numbered lanes) or buffer (odd-numbered lanes) were incubated with the indicated amounts of untagged M1 or an M1 mutant (‘prey’). After washing, bound proteins were eluted and analyzed by denaturing gel electrophoresis. (A) NEP binds to M1. (B) The C-terminal NEP domain binds to the N-terminal domain of M1. (C) Mutation of the NLS motif in M1 severely reduces binding to NEP. (D) Mutation of R101 and K102 residues in M1 reduces binding to NEP. (E) Mutation of K104 and R105 residues in M1 reduces binding to NEP. (F) Mutation of NEP residue Trp78 disrupts binding to M1. (G) Mutation of NEP residues Glu81 and Glu82 has little effect on binding to M1.

We next set out to localize the M1 binding site on NEP. We focused on residues which are solvent exposed in the structure of the NEP^59–116 dimer, reasoning that residues buried in the dimer were probably also buried in full-length NEP (and hence unable to mediate binding). Thus we examined the hydrophilic face of the helical hairpin, which is not involved in dimer formation, for possible binding epitopes. As described above, a conspicuous feature of this face is the prominently exposed Trp78 residue surrounded by a ring of glutamate residues (Figure 2D). This tryptophan is probably similarly exposed in full-length NEP, as near-UV circular dichroism (CD) and fluorescence spectroscopy have shown that the three aromatic residues in NEP are highly solvent accessible and able to rotate freely (Lommer and Luo, 2002). Therefore we constructed an NEP mutant in which Trp78 was replaced by a serine (NEP^W78S). We also made a double mutant in which residues Glu81 and Glu82 were substituted by alanines (NEP^E81A, E82A). These glutamates have their side chains located next to Trp78 but are not conserved in influenza B virus, where they are replaced by basic residues (Figure 2B and D). A pulldown assay shows that, whereas the double mutant retains significant affinity for M1, mutation of Trp78 severely disrupts binding activity (Figure 4F and G). Both mutant proteins appear to fold correctly, as they behave similarly to the wild-type species in gel filtration chromatography and CD spectroscopy (data not shown). We conclude that Trp78 is a major M1 binding epitope of NEP.

Given the large number of glutamate residues near Trp78, the interaction between NEP and the basic NLS of M1 probably has a significant electrostatic component. Tryptophan residues play an important role in NLS recognition by the nuclear import factor importin α, where they buttress lysine side chains of the NLS so that these can interact favourably with acidic residues of importin α (Conti et al., 1998; Conti and Kuriyan, 2000). Whether NEP recognizes the NLS motif of M1 by similar interactions must await determination of the structure of the M1/NEP complex.

Comparison of NEP proteins from type A and B influenza viruses

Although the NEP sequences from influenza A and B viruses share only 27% sequence identity (26% over the C-terminal domain), the chemical nature of residues is conserved at most positions, suggesting a common tertiary structure (Figure 2B). In particular, in the C-terminal domain the amphipathic nature of the helical hairpin and the highly charged character of helix C1 are preserved. Compared with influenza A, seven additional residues are present within the C-terminal domain of influenza B NEP, inserted between the C1 and C2 helices. These residues can be readily accommodated within the helical hairpin structure by a more elaborate interhelical loop or by extending one or both helices. Interestingly, the Trp78 residue implicated in M1 binding is replaced by a leucine in influenza B, suggesting that M1 recognition by NEP is somewhat different between the two viral types. Indeed, the M1 NLS motif in influenza A, (¹⁰¹RKLKR¹⁰⁵) differs from the corresponding motif in influenza B (RKMRR) at two positions. We speculate that the replacement of NEP residue Trp78 by the shorter Leu side chain is compensated by the replacement of M1 residues Leu103 and Lys104 by the longer Met and Arg side chains, permitting the mode of M1–NEP interaction to be essentially preserved between the two viral types.

Crystallization and structure determination

We obtained crystals of NEP^59–116 while attempting to crystallize a complex of NEP^59–116 bound to M1^1–165. A complex of the two proteins purified by Superdex 75 chromatography (Pharmacia) was concentrated to 20 mg/ml. Despite the presence of M1^1–165, crystals of the unbound NEP^59–116 species were obtained by hanging-drop vapour diffusion. The high ionic strength of the precipitant (0.4 M ammonium di-phosphate) probably caused dissociation of the complex.

Heavy-atom derivatives were prepared by soaking crystals in 5 mM K₂PtCl₄ for 2 h (derivative I) or 4 h (derivative II), or in 5 mM K₂OsCl₆ (2 h) in 0.4 M ammonium diphosphate. Data were collected at 100K using 20% glycerol as the cryoprotectant on an ADSC area detector at ESRF beamline ID14-2. Data were processed using MOSFLM (Leslie et al., 1992) and programs from the CCP4 suite (CCP4, 1994). Two heavy-atom sites were located in the platinum derivatives by SOLVE (Terwilliger and Berentzen, 1999), yielding an interpretable map after phase improvement with RESOLVE (Terwilliger, 2000) which automatically traced most of the main chain. Two low-occupancy Os sites were subsequently included for phasing at 2.8 Å resolution, and a final map was calculated to 2.6 Å resolution using non-crystallographic symmetry. Sixty-nine complete residues out of 116 were docked into the map by RESOLVE. The model was completed using O (Jones et al., 1991) and refined using REFMAC (Winn et al., 2001), treating the four helices of the dimer as independent TLS groups. The crystal packing was consistent with two possible choices of the biological dimer. However, the non-crystallographic dimer was easily assigned as the relevant one because of the large size and hydrophobic nature of its interface. Coordinates were deposited in the Protein Data Bank (accession No. 1PD3).

GTPase assays

Assays were performed as described (Bischoff et al., 1995; Kutay et al., 1997). First, 200 pmol of Ran were labelled with 30 nM [γ-³²P]GTP (5000 Ci/mmol). To measure substrate activities, 8 pmol of Ran [γ-³²P]GTP were incubated with 2 µM Crm1 and 0.001–20 µM of the tested substrate in a volume of 100 µl. After incubating for 15 min at 4°C, 10 µl aliquots of 0.1 µM Rna1p (the Schizosaccharomyces pombe RanGAP) were added to the mix and incubated for a further 2 min at 25°C. The reaction was stopped with 1 ml of activated charcoal and centrifuged (2 min at 12 000 g). Radioactivity in the supernatant was counted on a Wallac 1410 scintillation counter.