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letters A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein Marcus C.S. Lee1,2, Martin J. Scanlon3, David J. Craik3 and Marilyn A. Anderson1
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1School of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia. 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia. 3Centre for Drug Design and Development, University of Queensland, St. Lucia, Queensland 4072, Australia.
Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond linkage of Nand C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family. The reproductive and storage organs of many plants accumulate extremely high levels of serine proteinase inhibitors (PIs) for protection against predation and disease1. Regions of the female reproductive tissue of the ornamental tobacco, Nicotiana alata, amass PIs to levels of 20–30% of soluble protein2. These highly similar 6 kDa peptides inhibit either chymotrypsin or trypsin2, and are produced by proteolytic processing of a 43 kDa precursor protein, Na-PI2,3. Na-PI is composed of a signal peptide, six repeated regions each with a potential PI-reactive site, and a 25-residue C-terminal domain proposed to be a vacuolar targeting signal2,4 (Fig. 1d). Processing of the six-repeat Na-PI unexpectedly occurs at sites located within, rather than between, these repeated regions. Complete removal of the linker sequence (Glu-Glu-Lys-Lys-Asn) contained within each repeated region3, generates five contiguous inhibitors (C1, T1–T4) and two flanking peptides from the N- and C-termini (N-ter and C-ter, respectively; Fig. 1d). Here we show that the flanking peptides form a novel two-chain chymotrypsin inhibitor that can only be formed if Na-PI adopts a circular structure. Furthermore, circularization of the precursor protein is mediated by a change in domain folding during amplification from single to multiple repeats. A mechanism reminiscent of ‘domain swapping’ in oligomeric proteins5 is implicated in the evolution of the Pot II protein family of multidomain proteins.
A novel chymotrypsin inhibitor from N. alata flowers We purified PIs from stigmas by chymotrypsin affinity chromatography followed by reverse-phase (RP-) HPLC (Fig. 1a). The chymotrypsin inhibitor C1 and trypsin inhibitors T1–T4 (Fig. 1d) have been purified by this method3. However, a new protein (peak 1) appeared in extracts prepared without reducing agent (Fig. 1a). The protein in peak 1 is a chymotrypsin inhibitor (data not shown), which unlike C1–T4, migrated as a doublet 526
of less than 6 kDa on SDS–PAGE (Fig. 1a, inset). After treatment with 10 mM dithiothreitol (DTT) and 8 M urea, and reapplication to RP-HPLC, the protein in peak 1 separated into two peptides (Fig. 1b). N-terminal sequence and mass spectrometry data assigned the peak A and B peptides to the N-ter and C-ter fragments, respectively (Fig. 1c). The unreduced inhibitor in peak 1 (Fig. 1a) had masses of 5253.7 Da and 5138.1 Da, which correlated with the combined masses of the two peptides, less 8 Da lost by formation of four disulfide bonds. Thus, the protein in peak 1 corresponds to a novel two-chain inhibitor that we have designated as C2, composed of peptides processed from the N- and Ctermini of Na-PI (Fig. 1d). NMR structure determination of C2 We used a restraint set of 452 interproton distances inferred from NOE intensities, 14 backbone and eight side-chain dihedral angles to calculate the structures, and we obtained stereospecific assignments for five b-methylene pairs. The interproton distances from which redundancies based on the covalent geometry had been eliminated consisted of 149 intraresidue, 142 sequential, 57 medium and 104 long-range distances, including 54 long-range distance constraints between the two chains of C2. Of 12 slowly exchanging amide proton resonances observed, we unambiguously identified a corresponding acceptor for nine by inspection of the initial structures calculated in the absence of hydrogen bonding restraints. Subsequent rounds of structure calculations included restraints defining these hydrogen bonds. From the final round of structure calculations, we selected a family of 19 structures (from a total of 50) with the lowest energies and least residual violations of the experimental restraints to represent the solution structure of C2 (Table 1). The final 19 structures, when superimposed over the backbone heavy atoms (N, Ca, C) of the well-defined portion of the molecule (Fig. 2a), have no violations of distance or dihedral-angle restraints greater than 0.3 Å or 5.0°, respectively, they have good covalent geometry, as indicated by the small deviations from ideal bond lengths and bond angles, and have favorable nonbonded contacts indicated by the low values of the mean Lennard–Jones potential. Analysis of the family of structures in PROCHECK reveals that >90% of residues lie in either the most favored or additionally allowed regions of the plot. The major element of secondary structure is a triple-stranded antiparallel b-sheet. The N-ter peptide, Lys 1–Arg 18, contributes a single strand while the C-ter peptide, Asp 315–Glu 344, contributes the remaining two strands. The two peptides are tightly intermeshed, as evidenced by a total of 54 interchain NOE contacts. The reactive site (Leu 5–Asn 6) is situated close to the N-terminus of N-ter and constrained by two interchain disulfide bonds to C-ter (Fig. 2b). In general the structures of C2 are less well defined than those of the contiguous repeats. This is hardly surprising, as C2 is essentially identical to the single-chain chymotrypsin inhibitor C1 (ref. 6) after excision of a four-residue sequence from the reactive-site loop. Consequently, C2 has two N- and two C-termini, and the constituent peptides are covalently attached only through three disulfide bonds. In fact, no resonances are observable in the spectra for the N-terminal residue of N-ter or the three N-terminal residues of C-ter, an observation that may reflect additional mobility in these regions. The major difference between the structures of C2 and C1 is found in the central portion of the molecules, where the disulfide network links the well-defined region to the reactive site. In C1 this region forms a single turn of 310-helix, whereas in C2 a pair of overlapping b-turns is formed. nature structural biology • volume 6 number 6 • june 1999
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Fig. 1 Isolation of the two-chain inhibitor, C2. a, RP-HPLC profile of affinity-purified proteinase inhibitors, eluted with buffer B (dotted line). Peaks 2–5 were composed of PIs C1, T1–T4 as in (d). (inset) Silver-stained 15% SDS–PAGE of proteins (~4 mg) from peaks 1–5, respectively. b, RPHPLC profile of PIs treated with 10 mM DTT plus 8 M urea. Peptides from peak 1 (a) eluted as two peaks (A and B; solid line), while peptides from peak 3 eluted as a single peak (dashed line). c, N-terminal sequence and electrospray mass spectrometry data for peptides in peaks A and B (b). Unassigned residues (x) are probably cysteine. Sequence of the assigned peptides is shown in (e). d, Domain structure of Na-PI, showing the signal sequence (yellow), six tandem repeats each with a chymotrypsin (C) or trypsin (T) reactive site (red), and vacuolar targeting domain (green). Residues are numbered from the signal peptide cleavage site. Na-PI is processed at linker regions (orange) to release the individual inhibitors (C1, T1–T4) and the N-ter and C-ter fragments. e, Amino acid sequences of N-ter and C-ter aligned with the C1 sequence, shown as a representative of the other PIs. Only nine residues vary between all inhibitors. Reactive-site sequences are colored red.
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DRICTNCCAGTKGCKYFSDDGTFVCEGESDPRNPKACTLNCDPRIAYGVCPRS C125-77 C-ter315-344 DRICTNCCAGKKGCKYFSDDGTFICEGESE N-ter1-19 KACTLNCDPRIAYGVCPRS
Na-PI adopts a closed circular conformation As a consequence of the formation of C2, Na-PI must adopt an unusual closed circular structure composed of six PI domains (Fig. 3). This circular bracelet of inhibitors is clasped by the three disulfide linkages connecting the N-ter and C-ter fragments that form C2. Formation of disulfide bonds by these residues may be a critical event in the export of Na-PI from the endoplasmic reticulum (ER), as exposed thiols can retain unassembled or misfolded proteins in the ER7.
The proposed circular structure of Na-PI has some similarity to the structures of the neuraminidase subunit8 or the human regulator of chromosome condensation (RCC1)9, in that a b-sheet motif is repeated multiple times, with b-strands located at the N- and Ctermini contributing to one of the b-sheets. However, unlike these proteins, whose circular structures constitute a single functional domain, the six domains of Na-PI are distinct functional inhibitors, and circularization is necessary for the formation of the sixth inhibitor. Furthermore, these domains are then released by
a
b Fig. 2 NMR structure of C2. a, Solution structure of C2 showing backbone atoms (N, Ca and C) of N-ter (red) and C-ter (blue), with every fifth residue of N-ter and C-ter numbered. b, Ribbon representation of the C2 structure. The peptide chains are colored as in (a), except that the reactive site is green and the four disulfides are yellow in a ball-andstick representation. Figures were generated using MOLMOL18.
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Fig. 3 Model of Na-PI structure. The six PI domains are in different colors, with the N-ter and C-ter fragments of C2 indicated. The PI domains are modeled on the three-dimensional structures of the individual PIs (C2, this study; C1, ref. 6; T1–T4, ref. 14). The linker sequences are predicted to be hydrophilic and relatively unstructured. The C-terminal putative targeting domain has been omitted for clarity.
proteolysis in the linker regions. To our knowledge there is no precedent for such an arrangement in previously described proteins. Interestingly, Na-PI displays only partial protease inhibition activity, with only four out of a potential six reactive sites active against proteases3, and thus must undergo processing for full activity. Evolution of the multidomain precursor protein The bracelet structure adopted by Na-PI is a consequence of the transposed folding of an inhibitor domain across, rather than with-
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in, sequence repeats (Fig. 1d). However, the high identity between the six tandem repeats (79–100%) suggests that they arose from a single inhibitor sequence by gene duplication events. The ‘ancestral’ single-domain inhibitor may thus have adopted a fold different from the ‘present-day’ PI structure, such that it was accommodated within a single ‘sequence repeat’ unit (Fig. 4a). This ancestral PI would contain the N-ter and C-ter structural elements found in C2 and the single-chain inhibitors, but these elements would be reversed in order (that is, circularly permuted) relative to the native inhibitor (Fig. 4a). To determine whether a single ‘sequence repeat’ could encode a functional ancestral PI (aPI1), we produced the recombinant protein from the pET11a bacterial expression vector. The expressed protein was soluble and was recognized by polyclonal rabbit antisera generated to the native PIs; in addition, gel filtration-purified protein eluted as a single peak on RP-HPLC (data not shown), suggesting that it had folded into a single conformation. Furthermore, aPI1 displayed activity against chymotrypsin equivalent to the native C1 inhibitor (Fig. 4b). Double-repeat inhibitor reverts to present-day fold Finally, we wanted to determine if the presumed gene duplication event would produce a protein with inhibitor domains that still folded according to the sequence repeats, as observed for aPI1, or whether folding ‘reverted’ to the structural domains described for Na-PI. The recombinant double-repeat protein aPI2 (Fig. 4a) was expressed in an identical manner to aPI1, but was only partially soluble. Purified aPI2 inhibited chymotrypsin with a stoichiometry of one inhibitor to two protease molecules (Fig. 4b), indicating both reactive sites were functional. It is possible to propose two models for the structure of aPI2; in the first (Fig. 4c), the double-headed inhibitor could be generated by folding of inhibitors within repeats as tandemly linked aPI1 domains, joined by an alternative linker. In the second model (Fig. 4d), the PI domain folds across tandem repeats, with N- and C-terminal peptides covalently attached by disulfide bonding in a manner analogous to C2 in Na-PI. In this case, the conventional linker sequence Glu-GluLys-Lys-Asn would separate inhibitor domains. To distinguish between the two possibilities, aPI2 was digested with endoproteinase GluC and the products separated by RP-HPLC (data not shown) and analyzed by mass spectrometry. Mass spectrometry of the two RP-HPLC peaks produced (peak 1 and 2) is consistent with PI domains folding across Fig. 4 Ancestral PI. a, Domain structure of C1, C2, aPI1 and a two-repeat inhibitor (aPI2). Colors and numbering are identical to Fig. 1d. b, Inhibitory activity of aPI1, aPI2 and C1 against chymotrypsin (40 pmol). PI activity was expressed as a percentage of chymotrypsin activity remaining. c, Two models for aPI2 folding. Domain structure of aPI2, with the N-ter (N) and C-ter regions (C) separated by the linker region Glu-Glu-Lys-Lys-Asn (EEKKN, white bars). Model 1 shows aPI2 folding within repeats as two aPI1 domains in tandem, joined by an alternative linker sequence. d, Model 2 shows the inhibitor domain folding across two repeats, flanked by the linker sequence Glu-Glu-Lys-LysAsn (EEKKN), as occurs in Na-PI. A second inhibitor domain is generated by disulfide bonding of the equivalent N-ter (N) and C-ter (C') fragments. Arrows indicate potential endoproteinase GluC processing sites.
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Table 1 Statistics for the family of 19 C2 structures1 Mean pairwise r.m.s. deviations (Å)2 Backbone Heavy atom Arg10N-Val15N,Gly13C-Glu26C 0.54 ± 0.12 1.36 ± 0.23 Cys3N-Cys16N,Cys4C-Cys25C 1.51 ± 0.26 2.38 ± 0.33 Whole molecule 2.60 ± 0.48 3.84 ± 0.52 Mean r.m.s.d. from experimental restraints NOE (Å) 0.0142 ± 0.0030 Dihedral angles (°) 0.334 ± 0.266 Mean r.m.s.d. from idealized covalent geometry3 Bonds (Å) 0.0021 ± 0.00022 Angles (°) 0.724 ± 0.051 Impropers (°) 0.132 ± 0.022 Mean energies (kJ mol-1) ENOE4 2.97 ± 1.37 Edih4 0.14 ± 0.17 EL-J5 -150.5 ± 14.3 Ebond 2.0 ± 0.41 Eimproper 0.96 ± 0.35 Eangle 14.1 ± 3.2 Etotal -130.3 ± 16.8 The values in the table are given as mean ± standard deviation. Superscript numbers indicate the position of the residue in the sequence. Superscript characters N and C refer to the location of the position respective residues in the N-ter or C-ter peptide. 3Idealized geometry is defined by the CHARMm force field as implemented within X-PLOR. 4Force constants for the calculation of square-well potentials for the NOE and dihedralangle restraints were 50 kcal mol-1 Å-2 and 200 kcal mol-1 rad-2, respectively. 5The Lennard–Jones van der Waals energy was calculated with the CHARMm empirical energy function. 1 2
repeats (Fig. 4d). The observed masses of the peak 1 peptide (6230.9, 6359.7, 6488.8 Da) correspond to the two-chain inhibitor NC', while the peak 2 peptide (5800.3, 5929.3, 6058.3 Da) corresponds to the internal inhibitor CN' (Fig. 4d). Structure of the Pot II protein family Members of the Pot II family with two10,11, three12,13, four (unpublished results) and six2 repeats have been characterized within the Solanaceae. Here we show that two- and six-repeat proteins form a functional two-chain PI by adopting the ‘clasped bracelet’ fold, suggesting this general structure is adopted regardless of the number of intervening inhibitor repeats. To our knowledge, our results are the first confirmation that these two-chain inhibitors are formed by members of the Pot II family. A fascinating feature of the Pot II family is that the PI domain folds across tandem repeats, rather than within a repeat. This implies that a minimum of two sequence repeats are required to generate functional members of this family, apparently precluding amplification of an ancestral gene encoding a single PI as a mechanism by which the family evolved. However, our observation that a single-repeat unit (aPI1) can fold as a functional chymotrypsin inhibitor suggests that this may represent the ‘ancestral’ PI domain. Furthermore, when a gene-duplicated two-repeat protein (aPI2) is expressed, the PI domain ‘reverts’ to folding across repeats, as observed with the native proteins. Thus, the multidomain Pot II family may have evolved by gene duplication events from a singlerepeat unit encoding a functional PI. This amplification from single to multiple repeats generated a circular permutation of the PI sequence, allowing the inhibitor to fold across sequence repeats and the resulting N-ter and C-ter fragments to mediate circularization of the precursor proteins. Domain swapping The shift from intrarepeat associations between the N-ter and nature structural biology • volume 6 number 6 • june 1999
C-ter structural elements in aPI1 to interrepeat associations in aPI2 is reminiscent of the phenomenon of domain swapping described by Bennett et al. 5, a conformational rearrangement such that an interface between domains within a monomeric protein is disrupted and re-formed between corresponding domains on a different polypeptide chain5. So far, domain swapping has been used to describe oligomer formation, although a similar mechanism may equally apply to multidomain proteins on a single polypeptide chain. The first requirement is for a duplication of the ‘monomer’, in this case provided by the tandem PI repeats. Second, the interface between domains, or in this case the N-ter (N) and C-ter (C) structural elements, in the monomer is substituted by a similar interface in the ‘dimer’; that is, NC interface in aPI1 is converted to N–CN'–C' interface in aPI2 (Fig. 4d). This movement is mediated by the interdomain linker loops of sequence Glu-Glu-Lys-Lys-Asn. Unlike conventional domainswapped dimers, however, which may exist in their monomeric state, only one conformation of aPI2 has been detected. It is not known whether an ‘unswapped’ conformation of aPI2 (Fig. 4c) occurs transiently during folding in vitro.
Conclusion The advantage of producing multiple PIs from one precursor protein may lie in the ability to generate specificities against a broad spectrum of proteases and to promote efficient vacuolar sorting by utilizing a single targeting motif for every six inhibitors. Circularization of this remarkable molecule must involve unique mechanisms to achieve correct folding and disulfide bond formation, raising numerous questions about the molecules involved in protein folding within the plant secretory pathway. A large decrease in solvent-accessible surface area, resulting from circularization, may provide a driving force for the assembly of this novel, compact structure.
Methods Purification of mature PIs. Proteins were extracted from stigmas (15 g) in 100 mM Tris HCl, pH 8.0, and 10 mM EDTA and purified on chymotrypsin–Sepharose CL4B followed by RP-HPLC on a Brownlee RP300 C8 column (7 ´ 250 mm)2,3. Samples (~250 mg total protein) were applied in 0.1% (v/v) trifluoroacetic acid in H20 (buffer A) and eluted with 60% (v/v) acetonitrile, 0.089% (v/v) trifluoroacetic acid in H20 (buffer B). Chymotrypsin and trypsin inhibition assays. RP-HPLC purified PIs were lyophilized and resuspended in water for use in protease inhibition assays as described3. NMR spectroscopy. Samples contained ~0.7 mM peptide in either 90% H2O/10% 2H2O or 2H2O at pH 5.3 and pH 3.0. pH values are meter readings at 295 K, uncorrected for deuterium isotope effects. Oneand two-dimensional homonuclear spectra were recorded on a Bruker DRX750 spectrometer and processed as described6,14. Structure calculations. Distance constraints were derived from the intensity of crosspeaks in NOESY spectra recorded in 90% H2O /10% 2H2O and 2H2O with a mixing time of 200 ms. Initial structures were generated using DYANA15. Several rounds of structure calculation and assignment were performed to resolve ambiguities in the assignment of NOE crosspeaks. From the final assignment list, crosspeaks were classified as strong, medium, weak or very weak, and assigned corresponding upper bound distances of 2.7, 3.5, 5.0 and
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letters 6.0 Å, respectively. Pseudo-atom corrections were applied as necessary, and a further 0.5 Å was added to the upper bounds of restraints involving methyl protons. Dihedral-angle constraints were derived from 3JHNHa coupling constants and applied as follows: -120° ± 30° for 3JHNHa > 8 Hz; -60° ± 30° for 3J HNHa < 5 Hz. Additional f-angle restraints of -100° ± 80° were applied where the daN(i,i) NOE was clearly weaker than the daN(i,i + 1) NOE. Hydrogen bonds were defined by two pseudo-NOE constraints as follows: 1.58 < dHN < 2.30 Å; 1.58 < dON < 3.2 Å. Stereospecific assignments and c1 dihedral angle restraints were derived from 3JHaHb coupling constants measured from the ECOSY spectrum, together with dab (i,i) and dNb (i,i) NOE intensities16. The final set of structures was calculated using a dynamic simulatedannealing protocol in X-PLOR 3.117 as described6,14.The final 19 structures with the lowest overall energies that had no violations of distance restraints >0.3 Å or dihedral-angle restraints >5.0° were retained for analysis. Structures were visualized using the programs InsightII (Biosym) and MOLMOL18, and analyzed with PROMOTIF19 and PROCHECK20.
Bacterial expression and purification of aPI1 and aPI2. PCRamplified aPI1 and aPI2 regions were cloned into the pET11a vector (Novagen) for expression and cell lysis as described21. Purification was essentially as described for C2.
1. Richardson, M. Phytochemistry 16, 159–169 (1977). 2. Atkinson, A.H., Heath, R.L., Simpson, R.J., Clarke, A.E. & Anderson, M.A. Plant Cell 5, 203–213 (1993). 3. Heath, R.L. et al. Eur. J. Biochem. 230, 250–257 (1995). 4. Nielsen, K.J., Heath, R.L., Anderson, M.A. & Craik, D.J. Biochemistry 35, 369–378 (1996). 5. Bennett, M.J., Choe, S. & Eisenberg, D. Proc. Natl Acad Sci. USA 91, 3127–3131 (1994). 6. Nielsen, K.J., Heath, R.L., Anderson, M.A. & Craik, D.J. J. Mol. Biol. 242, 231–242 (1994). 7. Reddy, P., Sparvoli, A., Fagioli, C., Fassina, G. & Sitia, R. EMBO J. 15, 2077–2085 (1996). 8. Varghese, J.N., Laver, W.G. & Colman, P.M. Nature 303, 35–40 (1983). 9. Renault, L. et al. Nature 392, 97–101 (1998). 10. Bryant, J., Green, T.R., Gurusaddaiah, T. & Ryan, C.A. Biochemistry 15, 3418–3424 (1976). 11. Plunkett, G., Senear, D.F., Zuroske, G. & Ryan, C.A. Arch. Biochem. Biophys. 213,
463–472 (1982). 12. Taylor, B.H., Young, R.J. & Scheuring, C.S. Plant Mol. Biol. 23, 1005–1014 (1993). 13. Balandin, T., van der Does, C., Bellés Albert, J.M., Bol, J.F. & Linthorst, H.J.M. Plant Mol. Biol. 27, 1197–1204 (1995). 14 Nielsen, K.J., Heath, R.L., Anderson, M.A. & Craik, D.J. Biochemistry 34, 14304–14311 (1995). 15. Güntert, P., Mumenthaler, C. & Wüthrich, K. J. Mol. Biol. 273, 283–298 (1997). 16. Wagner, G. Prog. NMR Spectroscopy 22, 101–139 (1990). 17. Brünger, A.T. X-PLOR version 3.1. A system for X–ray crystallography and NMR. (Yale University Press, New Haven, Connecticut; 1992). 18. Koradi, R., Billeter, M. & Wüthrich, K. J. Mol. Graph. 14, 51–55 (1996). 19. Hutchinson, E.G. & Thornton, J.M. Protein Sci. 5, 212–220 (1996). 20. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. J. Appl. Crystallogr. 26, 283–291 (1993). 21. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning, a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1989).
A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site Damien Fleury1–3, Béatrice Barrère1,4, Thierry Bizebard2, Rod S. Daniels4, John J. Skehel4 and Marcel Knossow2 1These authors contributed equally to this work. 2Laboratoire d’Enzymologie et Biochimie Structurales, UPR 9063, CNRS, Bât. 34, CNRS, 91198 Gif-surYvette Cedex, France. 3Present address: L.C.M.,I.B.S.,41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France. 4National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. 530
Coordinates. The coordinates for the structure have been deposited in the Protein Data Bank (accession code 1QH2).
Acknowledgments We thank G.M. Neumann and R. Condron for electrospray mass spectrometry and Nterminal sequencing analysis, and E.A. Miller, T.J. Lithgow, N.J. Hoogenraad and A.E. Clarke for critical reading of the manuscript. This work was supported by a grant from the Australian Research Council. D.J.C. is an Australian Research Council Senior Fellow.
Correspondence should be addressed to M.A.A. email:
[email protected] Received 7 October, 1998; accepted 4 February, 1999.
These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during ‘antigenic drift.’ Hemagglutinin (HA) is the influenza virus glycoprotein that interacts with infectivity-neutralizing antibodies. As a consequence, amino acid substitutions that arise by mutations in the genes for HA over the years lead to escape of immune surveillance and recurrent epidemics—this process is called antigenic drift. The role of HA in influenza infection comprises two steps: (i) it mediates binding of the virus to its cellular receptors, sialic acid residues of glycoproteins or glycolipids, and (ii) following endocytosis, it mediates the fusion of viral and cellular membranes to permit entry of the genome–transcriptase complex into the cell. Hemagglutinin consists of a trimer of identical subunits, each made up of two disulfide-linked polypeptides, HA 1 and HA2. Structurally, each subunit consists of a membraneproximal helix-rich stem structure and a membrane-distal receptor binding globular domain1. The antigenicity of influenza isolates from outbreaks of disease and epidemics is regularly monitored to ensure the inclusion in vaccines of variants closely related to the circulating virus, and this surveillance is accompanied by sequencing of the genes for the HAs of representative isolates2. The antibody binding sites on HAs of viruses isolated since 1968 have been determined by locating sequence changes on the structure of the HA of influenza strain A/Aichi/2/68 (X31)3. The antigenic significance of amino acid substitutions occurring in these isolates has not been established directly, but these studies have been complemented by sequence analyses of the HAs of antigenic variants of X31 selected by growth in the presence of individual mononature structural biology • volume 6 number 6 • june 1999
