The novel crystal form of PR8 NS1 ED (W187A) shows the α/β fold common to all structures of NS1 ED (Bornholdt & Prasad, 2006
; Kerry et al.
). In contrast to the majority of NS1 structures, only one molecule of PR8 NS1 ED (W187A) was present within the asymmetric unit, although a strand–strand interface homologous to those observed for previous structures obtained using this construct was observed between symmetry-related monomers (Fig. 1
). As expected from its monomeric form, the helix–helix dimer present within all wild-type structures of NS1 was not observed in the crystal lattice (Fig. 1
). This is in agreement with previous structures obtained using this construct, which also lack the helix–helix dimer interface (Kerry et al.
). Therefore, since the PR8 NS1 ED W187A mutant is monomeric in vitro
(Kerry et al.
), it appears highly likely that the helix–helix dimer is the predominant interface for ED homodimerization. However, it is intriguing to observe that the strand–strand dimer is conserved among all three PR8 NS1 ED (W187A) structures that have now been solved. Additionally, this interface is also employed in two of the four wild-type PR8 NS1 ED structures [PDB entries 2gx9
(Bornholdt & Prasad, 2006
) and 3o9u
(Kerry et al.
)]. This is a remarkable coincidence, especially considering that the crystals leading to these five structures belonged to different space groups (P
22 and P
) and that conservation between mutant and wild-type structures is not observed for any other NS1 ED interface. Comparison of the strand–strand dimers indicates two distinct orientations of the monomers relative to one another (Supplementary Fig. 11
). Although one orientation predominates, the AF
dimers of 3o9u
adopt a slightly twisted arrangement, indicating that there may be some flexibility in the contacts formed at this interface. The partial conservation of the strand–strand packing interface indicates that while it may not allow ED dimerization, extension of the β-sheet is a possible method of interaction with cellular and viral binding partners. Interestingly, this interface is not observed in other structures of the NS1 ED, even when mutations preventing helix–helix dimerization are introduced [e.g.
W187Y (PDB entry 3kwi
) and W187A (PDB entry 3kwg
); Xia & Robertus, 2010
]. Therefore, it could be concluded that any functional properties of this interface may vary between influenza virus strains. Such strain-specificity has been observed for some functions of NS1, most notably interaction with CPSF30, which is associated with NS1s from H3N2 and H2N2 subtypes but not all isolates of the H1N1 subtype (Kuo et al.
; Hale, Steel et al.
). However, while the other NS1 ED solved from an H1N1 subtype (A/California/07/09; PDB entry 3m5r
; Center for Structural Genomics of Infectious Diseases, unpublished work) does not exhibit a strand–strand dimer, it does show antiparallel strand–strand interactions via
an alternative arrangement (Supplementary Fig. 21
). In structures of NS1 from PR8 the strand–strand dimer is formed by residues 88–91 of the two monomers interacting with one another to form a contiguous β-sheet; however, in the case of 3m5r
residues 80–85 of chain A
are sandwiched between residues 87–92 of chain G
and residues 86–89 of chain B
. This variant on the β-sheet augmentation theme suggests that such interactions may not be restricted by the sequences and arrangements observed in the strand–strand packing interface.
Figure 1 (a) Crystal structure of PR8 NS1 ED (W187A) described here (PDB entry 3rvc, shown in green). A strand–strand packing interface is formed with a symmetry-related molecule (shown in light grey). (b) Crystal-packing interactions formed between the (more ...)
Although the overall fold of the NS1 ED monomer is remarkably well conserved, comparison of all of the ED monomers solved to date indicates two regions of significant variation between the structures (Fig. 2). One region that appears to be capable of adopting a variety of positions is the 170-loop (residues 162–170), which is present at the interface between the ED and the iSH2 domain of p85β and has also been proposed to be a putative SH3-binding motif (Hale, Kerry et al.
; Shin et al.
). While the structure of this loop is always well ordered, the positions adopted vary between structures regardless of the strain or subtype (Fig. 2, left insert). Flexibility in this region may indicate a propensity for binding to a number of factors in addition to p85β, as several orientations may be required for different binding events.
Figure 2 Superposition of monomers of NS1 ED. The monomers are aligned with PR8 NS1 ED structure 2gx9_A. Monomers participating in strand–strand interactions are coloured red, whilst those not participating in such interactions are coloured blue. Inserts (more ...)
A second region of variance between NS1 ED monomers which has been observed previously (Hale, Barclay et al.
) is the N-terminus of the ED (up to residue 91) and the β-hairpin loop (140-loop) between the fourth and fifth β-strands (residues 135–143) (Fig. 2
, right insert). These residues appear to occupy one of two conformations, for which the A
chain of 2gx9
(Bornholdt & Prasad, 2006
) and the A
chain of 3d6r
(Hale, Barclay et al.
) could be considered to be archetypical structures. Interestingly, the position of this region does not appear to depend upon the sequence or strain of the ED crystallized, but rather on whether a strand–strand contact is formed within the crystal lattice. For example, the tertiary structure of the PR8 ED 3o9s_A
, which does not form a strand–strand dimer, bears more similarity to 3d6r_A
than to the previously characterized PR8 ED structure 2gx9_A
. Furthermore, a comparison of each ED monomer solved to date with these two archetypes (2gx9_A
) showed that while no monomer structure differed from either of the structures by more than 1.04 Å, all ED monomers involved in strand–strand contacts bore greater homology to 2gx9_A
than to 3d6r_A
(Table 2). Therefore, it appears to be likely that this orientation is induced by interactions at the strand–strand interface and may not exist outside of this context. In support of this view, examination of the NMR structure of an Udorn effector domain (PDB entry 2kkz
; Aramini et al.
) indicates that the 3d6r_A
conformation is adopted in all NMR states, while the 2gx9_A
conformation is not present. While the W187R mutation present within the monomer used to collect these NMR data is likely to disrupt ED dimerization, it is located within the helix–helix interface and is unlikely to influence any strand–strand interactions.
Analysis of NS1 ED-monomer homology
While it appears to be unlikely that the strand–strand dimer is the predominant ED homodimer, the partial conservation of this interface and the ability of contacts at this interface to induce conformational changes are interesting and may indicate that this surface may be utilized in other intermolecular interactions. Furthermore, the observation that other sequences can form a β-strand addition at this interface, as seen in 3m5r
, suggests that such β-sheet augmentations are unlikely to be restricted to the formation of a strand–strand dimer. Therefore, it is possible to envisage similar interactions existing between NS1 and one or more of the wide variety of factors that it is known to bind to.