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Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin
.I. Mol. J3iot. (1990) 216, 513-514
Crystallization and Preliminary X-ray Diffraction Studies of a Monoclonal Antibody Fab Fragment Specific for an Influenza Virus Haemagglutinin and of an Escape Mutant of that Haemagglutinin T. Bizebard192, Y. Mauguen’, F. Petek3, P. Rigolet’*’ J. J. Skehe14and M. Knossow1~2 1 Laboratoire Universite’ ’ Laboratoire
de Biologic Physicochimique, C.X.R.S., URA 1131 Paris Sud, Bat 433, 91405 Orsay Cedex, France
de Physique, Centre Pharmaceutique, C.N.R.S., 92296 Chatenay Malabry Cedex, France
3 Lahoratoire de Biologic Moldculaire des Interfe’rons, IRBC’, BP 8, 7 rue Guy Moquet, 94802 Viillejuif 4S.I.M.R.,
The Ridgeway,
Mill
Hill,
UPR 180
C.N.R.S., UPR 37 Cedex, France
London, ,YW7 IAA,
(Received 26 June 1990; accepted 16 August
U.K.
1990)
Preliminary crystallographic data are given for two molecules involved in the interaction bet’ween the humoral immune response and the influenza virus. These molecules are the Fah fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong l/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose struct’ure is known; they diffract to 3.4 .& resolution. Crystals of the Fab fragment are trigonal with space group P3,21 (or P3,21) and diffract t,o 2.6 A resolution. The unit cell dimensions are a = b = 98.9 8. c = 892 8. A native data set has been collected for both proteins.
Influenza virus has the outstanding characteristic t’o cause frequent epidemics of respiratory diseases. Each outbreak is caused by an antigenically distinct virus and the component primarily involved in antigenie variations is the haemagglutinin (HAT), a membrane-bound glycoprotein with which infectionneutralizing antibodies react. HA mutants that escape neutralization by monoclonal antibodies can be selected in the laboratory by growing influenza virus in the presence of a monoclonal antibody (Gerhard & Webster, 1978). Tn most cases, a single amino-acid mutant of HA is obtained and the observed mutations are similar to those of field isolates of viruses causing successive epidemics (Wiley et al.. 1981). In general, the contributions of particular amino acid substitutions to the mechanism of antigenic change are not known. There is strong evidence in one case that’ the site of amino acid substitution indicates the site of antibody binding: the threedimensional structure of an antigenie mutant of HA
t Abbreviation
(single amino acid change Glyl46-+Asp) has been determined and compared to that of wild-type HA to show that the only noticeable modification of HA was localized at t,he mutation site (Knossow et al., 1984). This result clearly indicates that the antibody binding site includes the mutation site: it does not precisely define which structural features are responsible for the decreased af?inity of the antibody. Tn order to gain more insight into the molecular mechanism that allows influenza viruses to escape neut’ralization by the humoral immune response, we have undertaken to determine the t’hreedimensional structure of the Fab fragment of a monoclonal antibody (HC19) directed against influenza haemagglutinin of strain X31 (Hong Kong l/68 (H3N2)), whose structure has been determined (Wilson et al.. 1981), along with the det’ermination of the structure of an HA mutant selected by this antibody (VA19, corresponding t,o the single amino acid change Serl57 + Leu). Crystals of VA19 have been obtained and are isomorphous to those of X31. A dat,a set to 3.4 A (1 A = til nm) has been collected.
used: HA. haemagglutinin. 513
0
1990 Academic
Press 1,imited
514
T. Bizebard et al.
We report here preliminary crystallographic data on the Fab fragment of antibody HC19. Monoclonal antibody HC19 (Mouse, IgG11) was produced as described by Fazekas de St Groth & Scheidegger (1980). The antibody was precipitated from ascitic fluid with ammonium sulphate at a final concentration of 1.86 M; this was followed by extensive dialysis versus phosphate buffered saline (pH 7.4) and by affinity chromatography on a Protein A-Sepharose CL4B column (Pharmacia) using recommended conditions (3 iw-NaCl, 1.5 M-glycine loading (pH 8.7)) to achieve efficient binding. The IgG was eluted in @l M-sodium citrate buffer (pH 5), dialysed in 10 mM-sodium phosphate buffer (pH 8.2) and chromatographed on a DEAE-Trisacryl (IBF) column, where it was eluted at 40 mM buffer concentration. The Fab fragment was prepared by papain digestion (4% papain (w/v), 1.25 mM-EDTA, 1.5 mm phosphate p-mercaptoethanol in @l M-sodium buffer (pH 7.3)). After six hours of incubation, papain was inactivated with @Ol M-iodoacetamide; the digest was dialysed in 10 mM-sodium phosphate (pH 8) and loaded onto a DEAE-Trisacryl column (IBF). The Fab fragment eluted in the flow-through and was thus separated from the Fc fragment which eluted at higher salt concentration. The Fab fragment was further purified by gel filtration on a Sephacryl SlOO-HR column (Pharmacia) equilibrated with phosphate buffered saline (pH 7). Finally, it was dialyzed against 0.15 M-Nacl, 0.1 y. (w/v) NaN, and concentrated to approximately 10 mg/ml by vacuum dialysis. When analysed by isoelectric focusing, this preparation contains three major species of approximate ~15.5, 6.0 and 6.3. The first one predominates in the crystals we usually obtain and it can be isolated by dialyzing the mixture in 10 mM-Tris buffer (pH 9), and chromatographying it on a &A-Trisacryl column (IBF); the species of interest elutes last by increasing the salt content of the buffer to 50 mM-NaCl. Electron micrographs taken under minimum dose conditions (Wrigley et al., 1983) of a solution containing the dissolved crystals of HC19 Fab fragment and X31 HA show mostly X31-Fab complexes, which is a confirmation of the functionality of the crystallizing species. Crystallization trials were carried out by the hanging drop procedure. Crystals were reproducibly obtained in drops consisting of a mixture of 4 ~1 of a protein solution (10 mg/ml HC19 Fab fragment in 0.15 M-NaCl, @l y. NaN3) and of 2 ,ul of a reservoir solution (1.2 M-K,HPO, (pH %8), 0.1 o/o NaN,); the drop was then equilibrated against reservoir solu-
Edited
tion at 18°C in a temperature-controlled room. Crystals grew to dimensions of 05 mm x 0.25 mm x 0.15 mm within a few weeks. X-ray precession photographs have shown the space group to be trigonal P3121 (or P3,21) with unit cell dimensions a = b = 98.9 8, c = 892 A. These unit cell dimensions, compared to those of other Fab fragments (Mariuzza et al., 1984), suggest that there is one molecule in the asymmetric unit. Crystals are stable for 36 hours at 4°C in the X-ray beam of a rotating anode (40 kV, 40 mA). X-ray reflections corresponding to 2.6 A resolution are visible on 1” oscillation photographs taken at the L.U.R.E. synchrotron. A native data set to 3 d has been collected and two potential heavy-atom derivatives and mercury tetrachloroplatinate (potassium phenylglyoxal (Monaco, 1978)) have been characterized. Determination of the structure has been and replacement using molecular undertaken isomorphous replacement methods. T. B. thanks the Fondation Marcel MBrieux for support,. We also gratefully acknowledge the use of data collection facilities at the EMBL outstation in Hamburg.
References Fazekas de St Groth, S. & Scheidegger, D. (1980). Production of Monoclonal Antibodies. Strategy and Tactics. J. Immunol. Methods, 35, 1-21. Gerhard, W. & Webster, R. G. (1978). Antigenic Drift in Influenza A Viruses. J. Exp. Med- 148, 383-392. Knossow, M., Daniels. R. S., Douglas, A. R., Skehel; J. ,J. & Wiley, D. (1. (1984). Three-dimensional Structure of an Antigenic Mutant of the Influenza Virus Haemagglutinin. Nature (London), 311, 678-680. Mariuzza, R. A., Amit, A. G., Boulot, G., Saludjian, P.. Saul, F. A., Tougard, P., Poljak, R. J., Conger. J.. Lamoyi, E. & Nisonoff, A. (1984). Crystallization of the Fab Fragment of Monoclonal Anti-p-azophenylarsonate Antibodies and Their Complexes with Haptens. J. Biol. Chem. 254, 5954-5958. Monaco, H. L. (1978). Ph.D. thesis, Harvard University. Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981). Structural Identification of the Antibody-binding Sites of Hong-Kong Influenza Haemagglutinin and Their Involvement in Antigenic Variation. Nature (London), 289, 373-378. Wilson, I. A., Skehel, J. J. & Wiley, 1). C. (1981). Structure of the Haemagglutinin Membrane Glycoprotein of Influenza Virus at 3 A Resolution. Nature (London), 389, 366-373. Wrigley, N. G.. Brown, E. & Chillingworth, R. K. (1983). Combining Accurate Defocus with Low-dose Imaging in High Resolution Electron Microscopy of Biological Material. J. Microac. 130, 225-232.
by R. Huber
Crystallization and Preliminary X-ray Diffraction Studies of a Monoclonal Antibody Fab Fragment Specific for an Influenza Virus Haemagglutinin and of an Escape Mutant of that Haemagglutinin T. Bizebard192, Y. Mauguen’, F. Petek3, P. Rigolet’*’ J. J. Skehe14and M. Knossow1~2 1 Laboratoire Universite’ ’ Laboratoire
de Biologic Physicochimique, C.X.R.S., URA 1131 Paris Sud, Bat 433, 91405 Orsay Cedex, France
de Physique, Centre Pharmaceutique, C.N.R.S., 92296 Chatenay Malabry Cedex, France
3 Lahoratoire de Biologic Moldculaire des Interfe’rons, IRBC’, BP 8, 7 rue Guy Moquet, 94802 Viillejuif 4S.I.M.R.,
The Ridgeway,
Mill
Hill,
UPR 180
C.N.R.S., UPR 37 Cedex, France
London, ,YW7 IAA,
(Received 26 June 1990; accepted 16 August
U.K.
1990)
Preliminary crystallographic data are given for two molecules involved in the interaction bet’ween the humoral immune response and the influenza virus. These molecules are the Fah fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong l/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose struct’ure is known; they diffract to 3.4 .& resolution. Crystals of the Fab fragment are trigonal with space group P3,21 (or P3,21) and diffract t,o 2.6 A resolution. The unit cell dimensions are a = b = 98.9 8. c = 892 8. A native data set has been collected for both proteins.
Influenza virus has the outstanding characteristic t’o cause frequent epidemics of respiratory diseases. Each outbreak is caused by an antigenically distinct virus and the component primarily involved in antigenie variations is the haemagglutinin (HAT), a membrane-bound glycoprotein with which infectionneutralizing antibodies react. HA mutants that escape neutralization by monoclonal antibodies can be selected in the laboratory by growing influenza virus in the presence of a monoclonal antibody (Gerhard & Webster, 1978). Tn most cases, a single amino-acid mutant of HA is obtained and the observed mutations are similar to those of field isolates of viruses causing successive epidemics (Wiley et al.. 1981). In general, the contributions of particular amino acid substitutions to the mechanism of antigenic change are not known. There is strong evidence in one case that’ the site of amino acid substitution indicates the site of antibody binding: the threedimensional structure of an antigenie mutant of HA
t Abbreviation
(single amino acid change Glyl46-+Asp) has been determined and compared to that of wild-type HA to show that the only noticeable modification of HA was localized at t,he mutation site (Knossow et al., 1984). This result clearly indicates that the antibody binding site includes the mutation site: it does not precisely define which structural features are responsible for the decreased af?inity of the antibody. Tn order to gain more insight into the molecular mechanism that allows influenza viruses to escape neut’ralization by the humoral immune response, we have undertaken to determine the t’hreedimensional structure of the Fab fragment of a monoclonal antibody (HC19) directed against influenza haemagglutinin of strain X31 (Hong Kong l/68 (H3N2)), whose structure has been determined (Wilson et al.. 1981), along with the det’ermination of the structure of an HA mutant selected by this antibody (VA19, corresponding t,o the single amino acid change Serl57 + Leu). Crystals of VA19 have been obtained and are isomorphous to those of X31. A dat,a set to 3.4 A (1 A = til nm) has been collected.
used: HA. haemagglutinin. 513
0
1990 Academic
Press 1,imited
514
T. Bizebard et al.
We report here preliminary crystallographic data on the Fab fragment of antibody HC19. Monoclonal antibody HC19 (Mouse, IgG11) was produced as described by Fazekas de St Groth & Scheidegger (1980). The antibody was precipitated from ascitic fluid with ammonium sulphate at a final concentration of 1.86 M; this was followed by extensive dialysis versus phosphate buffered saline (pH 7.4) and by affinity chromatography on a Protein A-Sepharose CL4B column (Pharmacia) using recommended conditions (3 iw-NaCl, 1.5 M-glycine loading (pH 8.7)) to achieve efficient binding. The IgG was eluted in @l M-sodium citrate buffer (pH 5), dialysed in 10 mM-sodium phosphate buffer (pH 8.2) and chromatographed on a DEAE-Trisacryl (IBF) column, where it was eluted at 40 mM buffer concentration. The Fab fragment was prepared by papain digestion (4% papain (w/v), 1.25 mM-EDTA, 1.5 mm phosphate p-mercaptoethanol in @l M-sodium buffer (pH 7.3)). After six hours of incubation, papain was inactivated with @Ol M-iodoacetamide; the digest was dialysed in 10 mM-sodium phosphate (pH 8) and loaded onto a DEAE-Trisacryl column (IBF). The Fab fragment eluted in the flow-through and was thus separated from the Fc fragment which eluted at higher salt concentration. The Fab fragment was further purified by gel filtration on a Sephacryl SlOO-HR column (Pharmacia) equilibrated with phosphate buffered saline (pH 7). Finally, it was dialyzed against 0.15 M-Nacl, 0.1 y. (w/v) NaN, and concentrated to approximately 10 mg/ml by vacuum dialysis. When analysed by isoelectric focusing, this preparation contains three major species of approximate ~15.5, 6.0 and 6.3. The first one predominates in the crystals we usually obtain and it can be isolated by dialyzing the mixture in 10 mM-Tris buffer (pH 9), and chromatographying it on a &A-Trisacryl column (IBF); the species of interest elutes last by increasing the salt content of the buffer to 50 mM-NaCl. Electron micrographs taken under minimum dose conditions (Wrigley et al., 1983) of a solution containing the dissolved crystals of HC19 Fab fragment and X31 HA show mostly X31-Fab complexes, which is a confirmation of the functionality of the crystallizing species. Crystallization trials were carried out by the hanging drop procedure. Crystals were reproducibly obtained in drops consisting of a mixture of 4 ~1 of a protein solution (10 mg/ml HC19 Fab fragment in 0.15 M-NaCl, @l y. NaN3) and of 2 ,ul of a reservoir solution (1.2 M-K,HPO, (pH %8), 0.1 o/o NaN,); the drop was then equilibrated against reservoir solu-
Edited
tion at 18°C in a temperature-controlled room. Crystals grew to dimensions of 05 mm x 0.25 mm x 0.15 mm within a few weeks. X-ray precession photographs have shown the space group to be trigonal P3121 (or P3,21) with unit cell dimensions a = b = 98.9 8, c = 892 A. These unit cell dimensions, compared to those of other Fab fragments (Mariuzza et al., 1984), suggest that there is one molecule in the asymmetric unit. Crystals are stable for 36 hours at 4°C in the X-ray beam of a rotating anode (40 kV, 40 mA). X-ray reflections corresponding to 2.6 A resolution are visible on 1” oscillation photographs taken at the L.U.R.E. synchrotron. A native data set to 3 d has been collected and two potential heavy-atom derivatives and mercury tetrachloroplatinate (potassium phenylglyoxal (Monaco, 1978)) have been characterized. Determination of the structure has been and replacement using molecular undertaken isomorphous replacement methods. T. B. thanks the Fondation Marcel MBrieux for support,. We also gratefully acknowledge the use of data collection facilities at the EMBL outstation in Hamburg.
References Fazekas de St Groth, S. & Scheidegger, D. (1980). Production of Monoclonal Antibodies. Strategy and Tactics. J. Immunol. Methods, 35, 1-21. Gerhard, W. & Webster, R. G. (1978). Antigenic Drift in Influenza A Viruses. J. Exp. Med- 148, 383-392. Knossow, M., Daniels. R. S., Douglas, A. R., Skehel; J. ,J. & Wiley, D. (1. (1984). Three-dimensional Structure of an Antigenic Mutant of the Influenza Virus Haemagglutinin. Nature (London), 311, 678-680. Mariuzza, R. A., Amit, A. G., Boulot, G., Saludjian, P.. Saul, F. A., Tougard, P., Poljak, R. J., Conger. J.. Lamoyi, E. & Nisonoff, A. (1984). Crystallization of the Fab Fragment of Monoclonal Anti-p-azophenylarsonate Antibodies and Their Complexes with Haptens. J. Biol. Chem. 254, 5954-5958. Monaco, H. L. (1978). Ph.D. thesis, Harvard University. Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981). Structural Identification of the Antibody-binding Sites of Hong-Kong Influenza Haemagglutinin and Their Involvement in Antigenic Variation. Nature (London), 289, 373-378. Wilson, I. A., Skehel, J. J. & Wiley, 1). C. (1981). Structure of the Haemagglutinin Membrane Glycoprotein of Influenza Virus at 3 A Resolution. Nature (London), 389, 366-373. Wrigley, N. G.. Brown, E. & Chillingworth, R. K. (1983). Combining Accurate Defocus with Low-dose Imaging in High Resolution Electron Microscopy of Biological Material. J. Microac. 130, 225-232.
by R. Huber