J. Mol. Biol. (1991) 221, 487497

Refined Atomic Structures of N9 Subtype Influenza Virus Neuraminidase and Escape Mutants W. R. Tulip’?, J. N. Varghese ‘, A. T. BakerIS, A. van Donkelaar’, W. G. Laver2, R. G. Webster3 and P. M. Colman1 ‘CSIRO Division of Biomolecular Engineering 343 Royal Parade, Parkville 3052, Australia 2John Curtin School of Medical Research Australian National University, Canberra 2601, Australia ‘St Jude Children$ Research Hospital Memphis, TN 38101, U.S.A. (Received

31 January

1991; accepted 13 May

1991)

The crystal structure of the N9 subtype neuraminidase of influenza virus was refined by simulated annealing and conventional techniques to an R-factor of 0.172 for data in the resolution range 60 to 2.2 A. The r.m.s. deviation from ideal values of bond lengths is 0014 A. The structure is similar to that of N2 subtype neuraminidase both in secondary structure elements and in their connections. The three-dimensional structures of several escape mutants of neuraminidase, selected with antineuraminidase monoclonal antibodies, are also reported. In every case, structural changes associated with the point mutation are confined to the mutation site or to residues that are spatially immediately adjacent to it. The failure of antisera to cross-react between N2 and N9 subtypes may be correlated with the absence of conserved, contiguous surface structures of area 700 A2 or more. Keywords:

influenza;

neuraminidase;

1. Introduction The influenza virus glycoprotein neuraminidase is one of two surface antigens attached to the viral membrane (for a recent review, see Krug, 1989). Selection pressure of host antibodies gives rise to amino acid sequence substitutions in the neuraminidase to the extent that sequences from year to year differ by a few per cent. Similar changes occur in the haemagglutinin, the other surface antigen. Occasionally, as a result of genetic reassortment, a major change in the structure of one of these molecules occurs and the characteristic of such a change is the failure of antisera to cross-react with the new antigen. Such an event defines a particular subtype of either neuraminidase or haemagglutinin. Amino acid sequence homologies between subtypes are of the order of 50%. Nine different neuraminidase subtypes have been characterized for type A t Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, U.S.A. $ Present address: LJniversity of Technology, Sydney _“007, Aust,ralia.

refined structure;

antigenicity;

mutants

influenza viruses, and two of them, Nl and N2, have been observed on viruses infecting humans. The remainder have been observed on avian, swine or equine viruses. The neuraminidase is a tetramer of polypeptides, approximately 469 amino residues in length (for a review, see Colman, 1989). Unlike soluble tetrameric enzymes characterized to date, which display dihedral 222 point group symmetry, neuraminidase possesses C4 symmetry. Glycosylation patterns vary from subtype to subtype. The three-dimensional structure of the N2 subtype neuraminidase has been determined (Varghese ef al.. 1983) and refined (Varghese & Colman, 1991) using material solubilized from viral membranes by Pronase digestion (Laver, 1978). Such a digest liberates so-called “heads” of neuraminidase, which comprise approximately residues 80 to 469, and which retain the and antigenic characteristics of enzymatic membrane-associated neuraminidase. The sequences of two N9 subtype neuraminidases have been determined (Air et al., 1985, 1987) and a preliminary report of the three-dimensional structure of one of them has appeared (Baker et al., 1987).

488

W. R. Tulip et al.

Roth N2, and N9 structures are based on a propeller fold, comprising six four-stranded antiparallel /?-sheets arranged as if on the blades of a propeller. The active site of the enzyme lies close to the propeller axis, which is approximately parallel to, and 30 A displaced from, the molecular 4-fold symmetry axis. Antigenic variation appears to occur predominantly on the various loops of the structure that connect the regular secondary structure elements (Colman et al., 1983). Field strain variation and the sites of mutation in antibody-selected variants are found in those loops. In contrast, the active site is an invariant structure, protected from antibody pressure by its size and possibly its invaginated shape (Colman et al., 1983). The shape and size aspects of functionally important conserved sites on antigens are believed to be important in rhinovirus (Rossmann, 1989) and foot-and-mouth disease virus (Acharya et al., 1989), respectively. The N9 structure is of particular interest for several reasons. Firstly, aggregated forms of this neuraminidase have the capacity to agglutinate red cells at 4°C (Laver et al., 1984). It has been suggested that this property of the N9 neuraminidase is attributable to a second sialic acid binding site, rather than to a modification of the enzyme active site, which might change the catalytic properties of the enzyme at low temperature (Webster et al., 1987). Secondly, the crystals that have been grown of this subtype are better ordered than their N2 counterparts and should provide a route to a more accurate structure than will be achievable by the N2 study (Varghese & Colman, 1991). Finally, we are seeking a structural correlate of the failure of antisera to cross-react between influenza subtype antigens. Here we describe the refinement of the N9 structure and of several variants of N9 selected in culture with monoclonal antibodies (Webster et al., 1987). These variants are able to grow in the presence of antibody, which inhibits the growth of wild-type virus, and are sometimes referred to as escape mutants. Typically they display single amino acid sequence changes with respect to wild-type, and are therefore of considerable interest in understanding mechanisms of immune recognition and antigenic variation. A comparison of the N9 and N2 structures is also presented.

K432N. These were selected with the antibodies NClO, NC24, NC31, 32/3 and NC34, respectively, as described by Webster et al. (1987). Data collection to 22 A was performed with an oscillation camera on a rotating anode generator. The crystal to film distance was 60 mm. Crystals were mounted along the [ 1 lo] axis and 1.2” oscillation photographs, with 02” overlap, were recorded with typical exposure times of 16 h. Approximately 23” of data were collected per dataset. Films were processed by the method and programs of Rossmann (1979). After the 2 films in each pack were scaled by a least-squares method (J. N. Varghese, unpublished results), data were loaded and scaled in PROTEIN (Steigemann, 1974) rejecting measurements for which (I - (I))/(I) is greater than 0.6. All 2F0 - FCmaps and difference maps were calculated in PROTEIN. Refinement of the structures was performed initially with PROLSQ (Hendrickson & Konnert, 1981) and subsequently with XPLOR (Brunger, 1988). Interactive graphics was done on a PS300 using software of Jones (1985). Prior to the calculation of 2F,, - F, electrondensity maps, calculated structure factors were corrected for solvent scattering by a least-squares method (Varghese & Colman, 1991). Consequently the lowresolution limit for the maps in the early stages of the refinement was extended from 6 A to 10 A. N9 mutants were analysed by difference Fourier techniques, initially using observed diffraction amplitudes to 2.9 A resolution of pairs of molecules, and finally using 2F, - FC amplitudes for the refined mutant structure in question. In each case independent refinement of the mutants was done with XPLOR. Analysis of solvent-accessible surfaces was by the method and programs of Connolly (1983) using extended van der Waals’ radii (Gelin & Karplus, 1979).

3. Results (a) Data collection A summary of the various X-ray diffraction data sets is given in Table 1. Data from native (i.e. wildtype N9) crystals are generally of poorer quality than those from the mutants, and for this reason one of the mutants (S37OL) was regarded as the reference structure for the purpose of refinement. The S37OL data set was collected from five crystals in 25 film packs, the range of internal R-factors of these film packs being 7-O to 9.7%, and the data set is 71.6% complete in the 6.0 to 2.2 A shell. (b) Refinement

2. Materials and Methods Isolation, purification and crystallization of the N9 virus influenza avian the from antigen A/tern/Australia/G70c/75 has been reported (Laver et al., 1984). The structure was solved by a combination of heavy-atom and Patterson search methods, using the N2 structure as a model (Baker et al., 1987). The crystals are cubic, space group 1432, a = 1851 A (1 A = 91 nm), and the molecular 4-fold axis coincides with the crystallographic tetrad. Crystallization of variants was as for wild-type N9, i.e. from 1.9 M-phosphate (pH 5.9). The variants studied are N329D (Asn329 to Asp), 1368R, A369D, S37OL and

A total of 14 rounds of PROLSQ and model building, in the final stages with isotropic temperature factor refinement, led to a model with R = @244 for data between 6 and 2.2 A resolution. The geometry of the model was poor (r.m.s.t devia.tions from ideal stereochemistry were for bonds O-031 A (target 0.02), for bond angle distances 9065 A (@03), for planarity 0018 A (0.02) and for chirality 0302 A3 (615)) and electron density maps t Abbreviations used: r.m.s., root-mean-square; NAG or GlcNAc, N-acetyl glucosamine: Man. mannose.

489

InJluenza Virus N9 Neuraminidase Structure

Table 1 Data collection statistics Number of observations

Dataset Wild-type Wild-type S37OL N329D 1368R A369D K432N

(a) (b)

29,387 45,520 75,224 104,979 54,110 52,953 59,403

Number of reflections

&(merge)

8500 18,424 19,112 17,915 15,374 14,449 14,599

6116 0130 9106 0121 0121 6119 0132

Completeness by sphere (%) 47.9 662 734 68.6 590 555 56.0

(2.5 (22 (2.2 (2.2 (2.2 (2.2 (2.2

A) A) A) A) A) A) A)

R,(merge) = Z(Zi - (Z))pZi, the sum being over all independent reflections. Mutants are as described in the text (S37OL being Ser370 to Leu370, etc.). The 2 wild-type sets are at resolutions of 2.5 A for (a) and 2.2 A for (b). Difference Fourier analyses refer to (a), and refinement refers to (b). Following refinement, a Luzzatti plot of the wild-type data set suggests that there may be systematic errors on this dataset, for which reason S37OL is described as the reference structure in this work.

were unconvincing in many places. We attribute these problems partly to the N2 model bias introduced at the start of the refinement. Indeed most of the segments where interpretation was difficult are those where the N2 and N9 structures differ most (see below). At this stage, molecular dynamics refinement (XPLOR) was being initiated on the structure of a complex between N9 and the Fab fragment of the anti-neuraminidase antibody NC41 (Tulip et al., unpublished results). From that work, parts of the structure, especially the double insertion in N9

relative to N2 at residue 412, were satisfactorily modelled. That segment, and other troublesome regions, were/updated from the N9-NC41 complex structure before initiating a full simulated annealing cycle of N9 refinement. In that cycle, the Pro431 peptide switched from bans to cis, a result which was confirmed later for the N9NC41 complex structure as well. Inspection of density maps and the resulting model showed that many substantial differences remained between the uncomplexed and complexed N9 structures. Such regions of N9 were set to

Table 2 XPLOR Cycle number A. Input data and settings Non-hydrogen atoms (no.) Protein Carbohydrate Water Residues Protein Carbohydrate Charges on side-chains turned off Method WA B. output Rin (%I Rou, (%) Final scale factor Fot.JFEaLE r.m.s. total co-ordinate shift (A) Main-chain Side-chain r.m.s. deviation from ideal stereochemistry Bond distance (A) Bond angle (“) Dihedral (“) Improper (“)

rejinement of S37OL

1

2

3

3056 0 0

3056 72 0

3067 111 90

387 0

387 6

388 9

4

3069

5

3069

111 94

111 94

388 9

388 9

no SA/Conv’l 243.150

SA/(!&v’l 253.510

Conv’l 200.000

Conv’l 200.000

Conv’l 200,000

32.5 21.1 9815

236 20.2 0804

21.2 17.7 0783

17.7 17.5 6785

17.9 17.2 0786

638 681

0.20 0.65

0.12 0.22

002 0.05

607 612

0020 39 28.9 1.7

9019 38 360 1.6

0.015 33 27.8 1.4

0015 32 27.8 1.4

0014 32 27.7 1.3

yes

yes

yes

The S37OL dataset was used throughout (Table 1) but only data in the range 6 to 22 A (17,687 reflections) were included. A calcium ion was introduced at cycle 4, and residue 370 was changed from Ser to Leu at the same stage. SA, Simulated annealing was performed only in cycles 1 and 2. Conv’l, Conjugate gradient minimization and/or restrained isotropic temperature factor refinement. WA, Weight on the crystallographic term in XPLOR. Ri, = EjlF,J - ~Fca,,~~/~jFor~ for starting model where Fobs and FEslsare the observed and calculated structure factors. R,,,, for output model.

W. R. Tulip et al.

490

dummy. This second cycle of simulated annealing on N9 resolved most of the discrepancies between N9 and N9-NC41. Three more cycles of energy refinement were run, during which a putative calcium ion site analogous to a similar feature in N2 (Varghese & Colman, 1991) was introduced, and an error in the register of the ten C-terminal residues 459 to 468 was corrected. Carbohydrate attached to Asn200, which has not been characterized chemically, had been omitted from the second cycle of simulated annealing. The resulting unbiased electron density confirmed the pattern of linkages that had been used in the N9-NC41 refinement. The monosaccharides and their linkages are GlcNAc 200A (B-1,4) GlcNAc 200B (p-1,4) Man 200C (a-1,3) Man 200D (a-1,2) Man 200E (cr1,2) Man 200F. The additional linkage at the branching mannose is Man 200C (a-1,6) Man 200G. Single GlcNAc residues were introduced at Asn86 and Asn146. This XPLOR refinement of S37OL is summarized in Table 2.

404

-120-

nn

P$-

?rA--_a-

- 180

/_

“A_“_

-120

-60

0

60

120

180

Phi (deg.)

Figure 1. Ramachandran plot of N9 (S37OL): (squares), residues other than glycine (crosses), regions from Ramachandran & Sasisekharan Ser404 lies just beyond the allowed regions.

glycines allowed (1968).

(c) Model quality The final R value is 0172 for all observed (i.e. I > 20(I)) data between 6 and 2.2 d. The geometry is tightly constrained with r.m.s. deviations on bond lengths of 0014 A and bond angles of 3.2” (Table 3). Final electron density maps (2F0 - F,) show the main-chain carbonyl oxygen atoms in density at the 1.0 (T level (here corrected for the electron density of the atom as described by Varghese & Colman (1991)), with the exception of residues 167 (0.6 0 corrected), 246 (@9), 248 (0.8), 293 (0.9), 331 (0.8). 358 (0.8) and 464 (0.8). On this scale. the mean main-chain atom density is nowhere lower than 1.5. Only seven side-chains remained as dummy features in the final refinement cycle (Arg82, Arg209, Lys261. Lys273, Glu414, Glu416 and Glu465). A number of electron density features on the uninterpreted. surface remain molecular Stereochemical considerations suggest’ that they are not water molecules, but they may represent solvent counterions. Ser404 is the only significant outlier on a Ramachandran plot (Fig. 1). A Luzzati (1952) plot (Fig. 2) suggests a positional standard deviation of 0.22 A. Water molecules have been introduced in chemi-

tally reasonable positions on the criterion that each was in a feature of electron density in a 2F, - F,

map contoured at 1.5 0, and that after energy refinement the feature remained at the 1.2 0 level. Only 93 are included in the present model, 39 fully buried from solvent (16 of these at the tetramer interface), 15 in the catalytic site, and a, further 39 distributed over the surface. The carbohydrate moiety at Asn200 is the best defined oligosaccharide in the structure (Fig. 3). The average temperature factors of all carbohydrat’e atoms is 28.1 A2 and for protein atoms 12.1 A2. (d) Mutants Difference electron density maps at a resolution of 2.9 a with amplitudes F,,,,,,,, - fi\370,_ and phases

0.40 0.35

~~ Ir 0.30 0.25 0.20

IO.7

g

.0.6

f

~0.5

i$

0.15

Table 3 ReJinement statistics for N9 and mutants

+ 0.2 ! O-I

R-V&X

structure S37OL N329D 1368R A369D K432S Wild-type Geometry

(b)

0.172 0.178 0.163 0160 0.167 0.209

Geometry

Resolution _ 6.2.2 6-23 6-2.3 6-2.3 6-2.3 6-2.2

values arc r.m.s. deviations

0014, 0.021, 0017, 0.017, 0016. @015,

--.-IOGO 002004006008010012014~16

3.2, 40, 3.5, 3.5, 3.4,

27.7 27.9 27.9 27.6 27.8

34,

280

from ideal bond lengths

(A), bond angles (deg.) and dihedral angles (deg.).

I / I 0180~20022024&026

_1 0.0

Stn~thetdllombdo

Figure 2. Plots of B-factor (filled squares) and datacompleteness by shell (open squares) on all reflections for which I > 2 a(l). WTSUS sin e/A, along with theoretical rurves (Luzzati. 1952) for the R-factor if all discrepancy is attributable to co-ordinate error. Data shown arr for t,he S37OL mutant’; the estimated mean positional error is 022 A.

Ir@uenza Virus N9 Neuraminidase Structure

491

Figure 3. Stereo view of electron density and model for the carbohydrate attached to Asn200. The 2F,, - F, map is contoured at 1.5 CT.

derived from XPLOR cycle 3 (Table 2) were analysed to arrive at a starting model for all mutants. (In this discussion we include wild-type when we refer to mutants.) In all cases it was apparent that the S37OL substitution in the reference structure had caused a displacement of the nearby Lys432. This conclusion was confirmed by similarly studying difference maps with coefficients Fmutant- F~32ana which showed no such perturbation of Lys432 except for the case where the mutant was S37OL. N329D is structurally closer to all other mutants than is S37OL and difference maps referring to N329D were used to determine starting models of the other structures. Following structural assignments from the difference maps, each structure was refined in XPLOR, including a 0.5 ps molecular dynamics refinement at 300 K followed by energy refinement and refinement of isotropic temperature factors. In the following, all map peaks are given in units of standard deviations (not normalized as described above). Difference maps are shown in Figure 4 with the structure of each mutant in continuous lines and that of the reference model (N329D or S37OL) in broken lines. (1) Wild-type: thelargest difference (FWT - FNszgD) density (Fig. 4(a)) peaks are + 6.0 and - 5.8 and are remote from the site of sequence difference (residue 329). The N329D substitution appears to have no effect on the structure, with the side-chains occupying identical positions. Refinement of the wild-type confirms this conclusion. structure Electron density in 2F, - F, map for the side-chain of Asp329 is connected only at the 1.0 CT level. Average temperature factors of side-chain atoms of residue 329 are 27.3 A2 in both S37OL and N329D, compared with 12.1 A2 for all protein atoms. (2) S37OL: the largest difference (FNa2aD J’s,,,,,) density (Fig. 4(b)) peaks are +7.7 (move-

ment of Lys432), +5.7 (possible water molecule on Lys432) and - 14.1 (Leu370). The largest uninterpretable peaks are +56 and - 7.3. Refinement confirms the interpretation. The position of Lys432 NZ in the two structures differs by 1.3 A due to displacement by the larger side-chain at position 370. (3) 1368R: the largest difference (F,368RF N329D) density (Fig. 4(c)) peaks are +10.5 (at Arg368), +6.4 (near residue 329): - 13.5 (at Ile368) and - 10-6 (at residue 329). The largest uninterpretable peaks are + 5.1 and - 6.3. Refinement supports the interpretation that the arginine sidechain at position 368 has displaced Asn329 from its position in the wild-type structure. Density in a 2F0 - Fc map describing the shifted position of Asn329 is only at level 1.0 CJfor the CR atom, but the amide group is at level 1.5. The 4rg side-chain at residue 368 is well defined. (4) A369D: a single peak of height + 11.2 is the only significant feature in the difference (FA369DF N329D)map (Fig. 4(d)). The largest uninterpretable features have peak height of + 4.8 and -5.8. The side-chain of Asp369 makes no interactions with other protein atoms, and occupies density at the 1.0 0 level only in a 2F,, - F, map. (5) K432N: the largest difference (FK432N F N329D)density (Fig. 4(e)) peaks are +7.7 (the Asn +5.6 (possibly a water molecule side-chain), -8.1, -8.0. -7.7, -7.4 (on attached to Asn432), 1~~~432) and -5.9 (at Pro431 0). The largest noise peaks are +5.2 and -5.9. Refinement confirms the interpretation, including the slight shift of the proline peptide, although the 2Fo - E’, density for this structure is poor. The Asn432 side-chain is not connected at the 1.0 (r level, although the head of the side-chain is in density. Of the mutants studied here, this dataset has the fewest observations (Table 1).

492

W.

R.Tulip

et al.

(b)

(d)

(el

Figure 4. Difference density (2.9 A resolution), with the refined models of the mutants (continuous thick lines) and reference structure (broken thick lines). Positive levels, broken contours; negative, dotted. Phases were derived from XPLOR cycle 3. (a) Wild-type (a)-N329D. Contour levels f3 Q. (b) N329D-S370L in the vicinity of residue 370. Contour levels +4 Q. (c) 1368R-N329D. Contour levels +4 6. (d) A369D-N329D. Contour levels +4 cr. (e) K432N-N329D. Contour levels’ +4 Q. The status of these structures shown in Table 3. (e) Antigenicity,

after refinement

is

surfaces and temperature factors

Figure 5 shows the variation of average residue temperature factor and exposed surface area (to a 1.7 A radius probe) for the neuraminidase tetramer. These parameters have a correlation coefficient of 0.418.

Also indicated in Figure 5 are the positions of mutation in ten escape mutants of neuraminidase selected with different monoclonal antibodies (Webster et al., 1987), and the residues of the binding sites for the NC41 and NC10 antibodies determined crystallographically (Tulip et al., unpublished results). Tainer et al. (1985) suggested that atomic mobility was correlated with antigenicity. In N9 neuraminidase, above average thermal parameters are seen not to be invariably associated with

Injluenza Virus N9 Neuraminiduse Structure

493

Table 4 Residues involved in lattice contacts in N9 (less than 4 tf ) From NA element

Residue

To symmetry operation

;::::

Arg284 Va1358

?iZ’axis

;;:

Pro33 1 Thr332 Va1333

{l/2-%,1/2-!/,1/2-s} Body-centring (&-fold axis)

B&,1

antibody binding sites (Fig. 5(b)), although it should be noted that the already high mobilities of two segments in the flsLol loop (318 to 353) are probably lower than in solution because of lattice contacts in the crystal, as detailed in Table 4. 150 % -

NA element

Residues L.eu263 Trp265 Tyr341 Pro328. Tyr341, Pro342, Gly343 Tyr341, Pro342

88:::

Similarly, large probe accessibility (Novotny et al., 1986) is not a rigorous measure of antibody binding sites (Fig. 5(c)). The segments involved in the epitopes recognized by NC41 and NC10 are all accessible to a 10 A probe, but some key residues in

. . .._........................................................................................................... I t---D---DC3----D---DC-D

1

(a)

pyl 100

-

8

& Lz 50 B 0) :: 0 ‘t u z -? 50 2 loo 100

150

?ii g

200

250

300

350

400 9 + : w m

E P

450 $! ‘: 71 (b)

Gi -z

E P

CC-N $8 Is>

P E m

T

NA residue number (N2 numbermg)

200

(c)

%! 150 cl : loo ‘t! 3 v)

D__DD__________(tDC__

50 0 100

150

200

250

300

B D

350 z P

400 c P E -El al

450 ?I D

G

Figure 5. Plots of various quantities of N9 (S37OL) wersus N2 residue number deletions in parentheses. Boxes indicate /?-sheet strands. The 3 carbohydrate residue to which they are attached: Asn86 (86 A), Asn146 (146 A), Asn200 (200 occurs in the tetrameric neuraminidase are the exposed surface area (upper) and subunit interfaces (lower). Calculations were performed using MS average temperature factor of each residue: sites of mut&ion in shown, as well as the epitopes (residues with buried surface area) (c) Accessibility to a large probe, as calculated using MS with a 10 mimic wild-type.

with insertions labelled A, B, etc., and moieties are marked according to the A-F). (a) Plotted for each residue as it the surface area buried at the subunit-

(Connolly, 1983) with a probe of radius 1.7 A. (b) The N9 (escape mutants) are marked with thick lines as in the tern N%NC41 and whale N%NClO complexes. A radius probe. Residue 370 was made into a serine to

494

W. R. Tulip et al.

those segments are not. The 366-372 loop, for example, is accessible at residues 368, 369 and 370, but the surface residues 367 and 372, which are contact residues with NC41, are inaccessible to a large probe.

(f) Sialic acid binding sites The result of soaking sialic acid into the N9 crystals has been to label only the enzyme active site. Details will be presented elsewhere, but there is still no direct evidence for bound sugar in the vicinity of the putative second sialic acid binding site (Webster et al., 1987).

(g) N%N9

comparison

Alignment of 216 CA atoms from the /?-sheet framework of N2 into N9 results in an r.m.s. deviation of 0.98 A. Omitting the 16 atom pairs with fit worse than 1.5 A (residues 96, 128, 162, 172, 173, 283, 286, 306, 310, 312, 356, 359, 360, 380, 381, 391) and realigning the structures improves the fit to 0.62 A. This transformation was used for subsequent comparisons. It reduces to Eulerian angle rotations of -9.4” around the Z-axis, 3.1” around the new position of the X axis and 0.2” around the

new position of the IT-axis. These values compare well with figures published earlier of - 90”, 2.7” and - 02” (Baker et aE., 1987). For all 385 (:A positions the fit is 1.18 A. The centre of gravity of the subunit in the N2 structure is 68 A closer to the 4-fold axis than its counterpart in N9. When the transformation is applied to the structure, ten segments have CA atoms with discrepancies of more than 2 A as shown in Figure 6. Mostly they occur in loop regions on the outer side and bottom of neuraminidase. and none of them impinges on the active site. The four sites of insertion or deletion in N9 with respect to N2 have been determined structurally on the basis of minimizing the discrepancy between equivalent CA positions as plotted in Figure 6. Those sites are 170A, 413A and B, 331 and 390. The sites originally assigned are 169A, 412A and B, 334 and 393 (Baker et al., 1987) and that sequence numbering has been retained here. The deletion in N9 at the end of the P5L,, loop (residue 390) results in a concerted repositioning of saccharides 2OOCand 200D. The most sequence-variable segment of the neuraminidase structure, both within (Colman, 1989) and between subtypes (Harley et al., 1989) is the loop ~&,. Its three-dimensional structure as judged by CA positions is also the most variable between N9 and N2. However, several structural features of this

%O 6.5 6.0 5.5

I

.I

5.0 45 s

40 g

3.5

x

3.0 2.5 2.0 I.5

I:

I.0 0.5 0.0 200

250

300

350

NA residue number (N2 numbermg)

400

450

8 $ 6

Figure 6. Plotted V,WWLSN2 numbering are the distances between equivalent CA atoms of N9 (S37OL) and N2 neuraminidase in the residue range 82 to 468, and between Cl atoms of the equivalent saccharidr units in the carbohydrate attached to Asn200 of a neighbouring NA subunit. For the purposes of this plot only, insertions and deletions in Pj9 with respect to Pj2 were chosen to minimize the discrepancy: insertions are 170A and 413A-413R. and deletions are 331, 390, and the C-terminal 469. P-Sheet strands of N9 and N2 are shown as boxes and homologous residues as thick lines.

InfEuenza Virus N9 Neuraminidase Structure

495

loop are conserved in all subtypes, belying its variability. Conserved sequences for the disulphide bond between residues 318 and 337 and the salt link between Asp330 and Arg364 are found in all known strains. The putative calcium ion site is also in this loop. Four of the ligands (293 0, 297 0, 324 ODl, 347 0) are common to N2 and N9. In N2, the fifth ligand is 345 0, but in N9 it is a water molecule, at the alternative position in the octahedral co-ordination sphere.

4.

Discussion

The neuraminidase polypeptides of influenza virus subtypes N2 (Varghese et al., 1983; Varghese & Colman, 1991) and N9 are similarly folded structures as expected from amino acid sequence homology of 47 y. and earlier structural studies (Baker et al., 1987). The similarity extends to the carbohydrate structure insofar as the best ordered oligosaccharide in both structures is that N-linked to Asn200 and non-covalently associated with a neighbouring subunit in the tetramer. The positioning of four saccharides (200 A to D) is similar in N9 and N2, but other saccharides are either in a different position (200 G) or have been built with a different chemical linkage (200 E). The similarity also extends generally to the dynamic structures of the proteins, at least to the extent that the profiles of temperature factors along the polypeptide chains are similar. A putative metal ion site, penta-coordinated with octahedral geometry, is also common to N2 and N9, although differences in the sequences and structures of the proteins in the neighbourhood of this site result in only four of the ligands being identical. As for N2 (Varghese & Colman, 1991), no evidence is found here for metal ion sites on the 4-fold symmetry axis where La’+ ions were observed to bind (Varghese et al., 1983). Small differences between N2 and N9 in the oligomer interfaces reported by Baker et al. (1987) are substantiated here. Numerous amino acid substitutions occur in residues at the oligomer interface, including the insertion around residue 169. The effect of these substitutions is twofold. The N9 and N2 subunits are rotated with respect to each other by some 3” across the tetramer interface and they are displaced to slightly differing radii from the 4fold axis. With respect to N2, N9 is opened out more at the bottom (i.e. near the stalk) than at the top. The 9” component of the transformation from N2 to N9 describes only differences of the crystal packing in the two structures. The positions of the five escape mutants in the tertiary structure of neuraminidase are shown schematically in Figure 7. 1368R, A369D, S37OL and K432N occur on the two short exposed loops p5L,, (368 to 371) and fi6LZ3 (431 to 437), which connect P-sheet strands. The loops are on the upper surface of neuraminidase and are positioned 5 to 10 A from the active site invagination. N329D is located on the side of the neuraminidase 16 A from the active

Figure 7. Schematic diagram of 1 of the 4 subunits of neuraminidase viewed down the molecular 4-fold rotation axis (bottom right). Arrows represent /?-sheet strands. For the escape mutants discussed in this paper, sites of mutation in N9 neuraminidase are numbered.

site, and is part of the convoluted loop flsLol (318 to 353). Escape mutants of rhinovirus map to four distinct regions and lie mostly in regions that form protrusions on the extreme exterior of the virus surface (Rossmann, 1989). The data on the single site escape mutants show, in all cases, only local structural changes associated with the site of the amino acid substitution or residues immediately adjacent to it. Two of the substitutions, 1368R and S37OL, cause a shift relative to wild-type in a nearby residue. Similar results have been described for the influenza H3 subtype haemagglutinin (Knossow et al., 1984) and N2 subtype neuraminidase (Varghese et al., 1988). The failure of monoclonal antibodies raised against the parent strain to bind to the selected mutant is directly attributable to these local changes. NC41 antibody has selected the escape mutants S367N and N400K (Air et al., 1989; Webster et al., 1987). In the case of the S37OL mutant selected by 3213 antibody, structural analysis of the N9NC41 Fab complex (Tulip et al., unpublished results) suggests that the replacement of Ser370 by the larger side-chain causes a simple steric interference with the formation of complex. In some cases, variants selected with one antibody continue to bind a second antibody, even though they form part of the binding surface for the second antibody. For example, N329D, selected by antibody NCIO, still binds NC41 antibody (Colman et al., 1987), and a study of the structure of that complex shows local side-chain rearrangement in the interface to accommodate the mutation (Tulip et al., unpublished results). Much larger rearrangements could occur as a result of an amino-acid substitution. Parry et al. (1990) have proposed a novel mechanism of antigenic variation in foot and mouth disease

496

W. R.Tulip et

al.

Figure 8. Space-filling shadowed models of N9 neuraminidase showing in different colours those residues identical in N9 and N2 (white) and residues of different amino-acid type (red). Left. The top surface of the neuraminidase is shown with the 4-fold axis at bottom right. Note the active site pocket towards the upper right which is conserved in sequences of all subtypes. It is surrounded by variable residues. Right. Side-view with the 4-fold axis at the middle rear. Antisera to one subtype could not cross-react with the other because there is no spatial epitope in common.

virus whereby a single-site mutation destabilizes the structure of a whole surface loop, which then folds into a completely different position from wild-type. Although there is no formal objection to such a mechanism operating, the evidence remains indirect, particularly on the issue of whether or not antibody can directly see the site of the mutation (Pqry et aE., 1990). This study has shed no further light on the haemagglutinating activity of N9 neuraminidase (Laver et aE., 1984). Although the haemagglutinating properties of wild-type and variant N9 molecules implicates a site near residues 369, 370, 372 and 400, that binding, if it exists, is weaker than can be detected under the present soaking conditions. A recent soaking experiment and data collection at 8°C has also failed to produce direct evidence for binding elsewhere than at the enzyme active site (J. N. Varghese & P. M. Colman, unpublished results). The failure of antisera to N2 or N9 subtype neuraminidases to cross-react with the heterologous subtype may have its structural origins in the absence of conserved surface regions of the order of based on structural studies of 700 A2 which, antigen-antibody interactions (Amit et al., 1986; Colman et al., 1987; Colman, 1988) are an anticipated requirement for such cross-reaction. This result is highlighted in Figure 8. The surface area of the invaginated conserved active site (Fig. 7(a)) is about 600 d2 and it appears that this site is privileged because antibody is unable to see it at the exclusion of catalytically nonessential and strain-variable amino acids (Colman et al., 1983).

We thank Paul Davis for assistance with computing. This work was supported by an Australian Postgraduate Research Award and a CSIRO Institute of Industrial Technologies Postgraduate Studentship to W.R.T. and bJ U.S. Public Health Service research grants AI-21659 (to P.M.C.) and AI-08831 (to R.G.W.) from the National Institutes of Health. This work was supported in part by Cancer Centre Support (CORE) grant CA-21765 and by American Lebanese Syrian Associated Charities to R.G.W. Deposition: co-ordinates, and the observed and calculated structure factors of the six N9 neuraminidase structures presented here have been deposited (entry numbers lNN9 to 6NN9) in the Brookhaven Protein Data Bank to be released as of 1 July 1991.

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Edited by R. Huber