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Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides. The distinguishing characteristics of BH3 peptides capable of directly activating Bax were also elucidated. Here we identify specific BH3 peptides capable of "unlatching" Bak and describe structural insights into Bak activation and oligomerization. Crystal structures and crosslinking experiments demonstrate that Bak undergoes a conformational change similar to that of Bax upon activation. A structure of the Bak core domain dimer provides a high-resolution image of this key intermediate in the pore-forming oligomer. Our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to certain BH3 peptides.

SummaryIn stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2–α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other’s surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

SummaryThe prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2–regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2–like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with “BH3 mimetic” drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

The Bcl-2 family regulates induction of apoptosis at the mitochondria. Essential to this regulation are the interactions between Bcl-2 family members, which are mediated by Bcl-2 homology (BH) domains. Vaccinia virus F1L is a unique inhibitor of apoptosis that lacks significant sequence similarity with the Bcl-2 family and does not contain obvious BH domains. Despite this, F1L inhibits cytochrome c release from mitochondria by preventing Bak and Bax activation. Although F1L constitutively interacts with Bak to prevent Bak activation, the precise mechanism of this interaction remains elusive. We have identified highly divergent BH domains in F1L that were verified by the recent crystal structure of F1L (Kvansakul, M., Yang, H., Fairlie, W. D., Czabotar, P. E., Fischer, S. F., Perugini, M. A., Huang, D. C., and Colman, P. M. (2008) Cell Death Differ. 15, 1564-1571). Here we show that F1L required these BH domains to interact with ectopically expressed and endogenous Bak. The interaction between F1L and Bak was conserved across species, and both F1L and the cellular antiapoptotic protein Mcl-1 required the Bak BH3 domain for interaction. Moreover, F1L replaced Mcl-1 during infection, as the Bak x Mcl-1 complex was disrupted during vaccinia virus infection. In contrast to UV irradiation, vaccinia virus infection did not result in rapid degradation of Mcl-1, consistent with our observation that vaccinia virus did not initiate a DNA damage response. Additionally, Mcl-1 expression prevented Bak activation and apoptosis during infection with a proapoptotic vaccinia virus devoid of F1L. Our data suggest that F1L replaces the antiapoptotic activity of Mcl-1 during vaccinia virus infection by interacting with Bak using highly divergent BH domains.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S(2) specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

Overexpression of members of the BCL-2 family of pro-survival proteins is commonly associated with unfavourable pathogenesis in cancer. The convergence of cytotoxic stress signals on the extended BCL-2 protein family provides the biological rationale for directly targeting this family to induce apoptotic cell death. Recently, several compounds have been described that inhibit the interaction between BCL-2 family members and their natural ligand, a helical peptide sequence known as the BH3 domain. Here, we review preclinical and clinical data on these compounds, and recommend four criteria that define antagonists of the BCL-2 protein family.

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2–like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)–only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.

Summary Many viruses express antiapoptotic proteins to counter host defense mechanisms that would otherwise trigger the rapid clearance of infected cells. For example, adenoviruses and some γ-herpesviruses express homologs of prosurvival Bcl-2 to subvert the host's apoptotic machinery. Myxoma virus, a double-stranded DNA virus of the pox family, harbors antiapoptotic M11L, its virulence factor. Analysis of its three-dimensional structure reveals that despite lacking any primary sequence similarity to Bcl-2, it adopts a virtually identical protein fold. This allows it to associate with BH3 domains, especially those of Bax and Bak. We found that M11L acts primarily by sequestering Bax and Bak, thereby blocking the killing action of these essential cell-death mediators. These findings expand the family of protein sequences that act like Bcl-2 to block apoptosis and support the conclusion that the prosurvival action of these proteins critically depends on their ability to bind and antagonize Bax and/or Bak.

The three-dimensional structure of the haemagglutinin-neuraminidase (HN) from a human parainfluenza virus is described at ca 2.0 A resolution, both in native form and in complex with three substrate analogues. In support of earlier work on the structure of the homologous protein from the avian pathogen Newcastle disease virus (NDV), we observe a dimer of β-propellers and find no evidence for spatially separated sites performing the receptor-binding and neuraminidase functions of the protein. As with the NDV HN, the active site of the HN of parainfluenza viruses is structurally flexible, suggesting that it may be able to switch between a receptor-binding state and a catalytic state. However, in contrast to the NDV structures, we observe no ligand-induced structural changes that extend beyond the active site and modify the dimer interface.

The fusion of viral membranes with target-cell membranes is an essential step in the entry of enveloped viruses into cells, and recent X-ray structures of paramyxoviral envelope proteins have provided new insights into protein-mediated plasma-membrane fusion. Here, we review our understanding of the structural transitions that are involved in this fusion pathway, compare it to our understanding of influenza virus membrane fusion, and discuss the implications for retroviral membrane fusion.

The fusion protein of respiratory syncytial virus (RSV-F) is responsible for fusion of virion with host cells and infection of neighbouring cells through the formation of syncytia. A three-dimensional model structure of RSV-F was derived by homology modelling from the structure of the equivalent protein in Newcastle disease virus (NDV). Despite very low sequence homology between the two structures, most features of the model appear to have high credibility, although a few small regions in RSV-F whose secondary structure is predicted to be different to that in NDV are likely to be poorly modelled. The organization of individual residues identified in escape mutants against monoclonal antibodies correlates well with known antigenic sites. The location of residues involved in point mutations in several drug-resistant variants is also examined.

2,3-didehydro-2-deoxy-N:-acetylneuraminic acid (DANA) is a transition state analog inhibitor of influenza virus neuraminidase (NA). Replacement of the hydroxyl at the C9 position in DANA and 4-amino-DANA with an amine group, with the intention of taking advantage of an increased electrostatic interaction with a conserved acidic group in the active site to improve inhibitor binding, significantly reduces the inhibitor activity of both compounds. The three-dimensional X-ray structure of the complexes of these ligands and NA was obtained to 1.4 A resolution and showed that both ligands bind isosterically to DANA. Analysis of the geometry of the ammonium at the C4 position indicates that Glu119 may be neutral when these ligands bind. A computational analysis of the binding energies indicates that the substitution is successful in increasing the energy of interaction; however, the gains that are made are not sufficient to overcome the energy that is required to desolvate that part of the ligand that comes in contact with the protein.

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

The structure of the trypanosomal trans-sialidase reveals a canonical sialidase catalytic site elaborated with a conformational switch that creates an adjacent binding pocket for lactose.

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

Recent studies of the structural basis of antigenic variation in influenza virus neuraminidase and of the structure of neuraminidase-antibody complexes are summarized

Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-x(L), and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-x(L) and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-x(L), a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.

Zanamivir is the first of two registered neuraminidase inhibitors for the treatment and prophylaxis of influenza. Relenza®, an orally inhaled powder form of zanamivir, is currently approved in 19 countries for treatment, and in two for prophylaxis. Relenza reduces the time to alleviation of symptoms by 1 to 2 days in the influenza-positive population, if taken within 48 h of symptom onset, and in prophylaxis in family settings, it confers an 80% reduction in the odds of contracting influenza. The resistance profile of zanamivir is encouraging in the sense that there are still no reports of patients on acute therapy shedding drug-resistant virus. However, patient uptake of the inhaled drug has been insufficient to conclude that drug resistance will not be an issue in the future. All zanamivir-resistant variants selected in the laboratory so far have diminished viability.

Two potent inhibitors based on the crystal structure of influenza virus sialidase have been designed. These compounds are effective inhibitors not only of the enzyme, but also of the virus in cell culture and in animal models. The results provide an example of the power of rational, computer-assisted drug design, as well as indicating significant progress in the development of a new therapeutic or prophylactic treatment for influenza infection.

The structure of a χ-type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur. J. Biochem. 45, 513 (1974)). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 a. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.

A description is given of a computer controlled rotating drum film scanner suitable for the measurement of X-ray diffraction photographs. The advantages of such a densitometer for protein crystallography are discussed. By measuring integrated densities 'on line' and taking advantage of the high positional accuracy of the scanner, a very satisfactory combination of speed, accuracy and flexibility can be achieved. It is found that commonly used X-ray films are significantly nonlinear even at optical densities less than 1.2 but by using a parabolic modification of the integrated densities the usable density range can be extended to optical densities of at least 2.5.

SummaryThe immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.

We have recently reported the X-ray crystal structure of a fragment of the fusion protein (F) of Newcastle disease virus (NDV). This work describes the methodology involved in the production and crystallization of that protein in recombinant form. The full-length cDNA of NDV-F was cloned and the ectodomain expressed in both CHO-K1 and Lec-3.2.8.1 cells. The recombinant protein, secreted as a single-chain polypeptide F0′, was purified using a c-myc antibody affinity column followed by gel filtration chromatography. Electron microscopic imaging showed the F0′ product to consist of unaggregated club-shaped particles. Trypsin treatment of F0′ could be used to produce disulfide-linked F2 and F1′ chains. However, imaging revealed extensive rosette-like aggregation of the trypsin-treated material, indicative of a conformational change. Only the non-trypsin-treated product was thus suitable for crystallization and two crystal forms were obtained, diffracting to ca. 3.5 and 4.0 Å, respectively. Both crystal forms were used in the structure determination.

Phaseolin, the main reserve globulin in seeds of the French bean (Phaseolus vulgaris L.), has been purified for sedimentation equilibrium analysis. The major protomer at neutral pH has a molecular weight of 150 000±5 000 which associates to a tetrameric form of molecular weight 596 000±20 000 at pH 4.5. The trimeric nature of the protomer is apparent from protein crosslinking experiments. Initial structural studies were made using optical rotatory dispersion and circular dichroism measurements. Two different crystal forms of the protein have been grown and characterised by X-ray diffraction; one of these forms appears suitable for high resolution X-ray analysis.

The low-resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X-ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point-group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry. Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry.

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.