Immumlogy and Cell Biology (1992) 70, 209-214
Structural basis of antigenic variation: Studies of influenza virus neuraminidase p. M. COLMAN CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia Summary Recent studies of the structural basis of antigenic variation in influenza virus neuraminidase and of the structure of neuraminidase-antibody complexes are summarized.
Introduction The immune system has a remarkable capacity to generate a large number of binding specificities among the family of proteins which act as receptors for antigen. The terms 'binding' and 'specificity' are commonly used in biology to describe the characteristics of molecular interactions, but the structural principles which determine the response of two molecules to each other are both complex and subtle. Studies of antibody-anti gen interactions in solution have illustrated both of these characteristics. The complexity of the phenomenon is highlighted by the capacity of antibodies to cross-react with proteins unrelated to the immunizing antigen, and its subtlety is underscored by the failure of monoclonal antibodies to bind to single site variants of antigen in which the amino acid substitution is conservative. Studies by X-ray crystallography of the three-dimensional structure of influenza virus neuraminidase and of its complex with monoclonal antibodies have now revealed some of the structural principles which govern the formation of complexes. This paper summarizes recent work.
Antigenic variation in influenza viruses The two envelope-associated antigens of type A influenza virus, neuraminidase and haemagglutinin, display structural variation from strain to strain, which has previously been reviewed by Krug.' Antigenic subtypes of both the neuraminidase and haemagglutinin are defined by the capacity ot antisera to cross-react with other members of the group, and within such groups the amino acid sequences of the antigens differ by about 10-20%. Sequence differences between subtypes are of the order of 50%. Nine subtypes of neuraminidase have been identified and the three-dimensional structures of two of them, N2~'^ and N9'* have been determined by X-ray crystallography. Within the soluble 'head' portion of the molecule which can be cut from the viral membrane with enzyme, the N2 and N9 sequences are 48% identical. In two places in the sequence N9 has insertions with respect to N2 and in two other places it has single residue deletions. The three-dimensional structures of the two antigens are very similar at the level of the polypeptide fold, each structure displaying a
Correspondence: P. M. Colman, CSIRO Division of Biomolecular Engineering. 343 Royal Parade, Parkville, Vic. 3052, Australia. Presented at the 4th Frank and Bobbie Fenner Conference in Medical Research on Getietk Variation and Selection in Microbiology and Immunology, held on 6-8 November 1991 at the John Curtin School of Medical Research, Canberra, Australia. Accepted for publication 6 November 1991.
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P. M Colman
Fig. 1. Schematic drawing of the neuraminidase monomer. The N-terminal residue of the enzyme solubilized neuraminidase is near residue 80 and is on the under-side in this view, presumably the membrane-proximal side of the molecule. The chain folds in six topologically identical fourstranded P-sheets. (Reproduced from Varghese and Colman.") six-bladed p sheet propeller motif (Fig. 1). The region of the structure in which the amino acid sequence is most variable, both within and between subtypes, is the segment linking p sheets 4 and 5, residues 320-350. There also the three-dimensional structures of N2 and N9 are least similar.'* The failure of antisera to cross-react between N2 and N9 structures can be understood in terms of what is now known about the size of a binding site for antibody on an antigen. Approximately 700 A^ of surface area is usually abstracted from solvent when an antibody-antigen complex forms. Figure 2 shows two orthogonal views of the N9 structure,** colour coded to indicate regions of amino acid sequence identity with N2. The largest such contiguous region on the surface of the molecule is of the order of 500 A^ seen in the upper right ofthe left-hand panel. This is the enzyme active site and it is probably too small to bind to an antibody. In contrast, field strain variation within subtypes results in large areas of exposed surface being unmodified from year to year, and these are presumably the basis of crossreaction by antisera. For example, among the
known sequences of N2 strain.^ the neuraminidase is altered at some 27 residues between the years 1957 and 1968 and at a further 16 positions between 1968 and 1972 (Fig. 3). Large regions of the surface remain conserved during these periods and they are presumably the targets of cross-reacting antibodies. The foregoing discussion has all related to field strain variation ofthe antigens. There are now many examples of laboratory variants, the most interesting of which have been selected with monoclonal antibodies.'''' Typically these variants differ from wild type by a single amino acid substitution. That substitution suffices effectively to abolish the binding of the monoclonal antibody to the wild type antigen against which it is raised. The types of substitution seen in these experiments include most ofthe amino acid exchanges possible by single base substitution (Fig. 4). A number of three-dimensional structures of monoclonal variants have now been studied for the haemagglutinin^ and neuraminidase,^'^ and in all cases to date they show only local structural perturbation at, or immediately adjacent to, the site of the amino acid substitution.
Neuratninidase-antibody complexes Two monoclonal antibodies to influenza vims N9 neuraminidase, NC41 and NCIO, have been studied by crystallography as complexes with the antigen.^ '^ Forthe NC41 complex the central findings are as follows. Five linearly discontinuous surface loops of the neuraminidase contact the antibody through five of its six complementarity-determining regions (CDR). Nineteen amino acids on the antigen are in contact with 17 on the antibody. The surface areas buried by antigen and antibody as a consequence of complex formation are each 880 A (Fig. 5). There are 12 hydrogen bonds and three ion pairs in the interface. These observations are similar to those of others working on the lysozyme-antibody complexes (reviewed by Davies and Padlan'^). In the case ofthe neuraminidase complex, the interaction is somewhat more extensive.
Structural basis of antigenic variation
211
Fig. 2. Space-filling shadowed models of N9 neurarainidase showing in different colours those residues identical in N9 and N2 (white) and residues of different amino acid type (red). Left. The top surface ofthe neuraminidase is shown with the four-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 four-fold axis at the middle rear. Antisera to one subtype could not cross-react with the other because there is no common spatial epitope ofthe required size (approximately 800 A^). (Reproduced from Tulip etal.'*)
The NCIO structure is not yet complete, but it is quite clear that it binds an epitope overlapping that of NC41. The detailed analysis of that structure will demonstrate how two different antibodies bind the same structures on an antigen. This phenomenon has a parallel in anti-idiotypy, where an antibody raised to an immunizing antigen can itself be used to raise an antibody response directed at its CDR. The resulting anti-idiotypic antibody and the initial antigen do not necessarily share obvious structural characteristics, although they both bind the primary antibody. One antibodyanti-idiotypic antibody complex structure has
been studied by X-ray crystallography,^** demonstrating that the antigen, lysozyme in that case, and the anti-idiotypic antibody have little in common. The anti-neuraminidase antibodies have not yet been studied in their unliganded form to permit a comparison of the bound and free structures.
Antibody structure and binding properties The binding site for antigen on antibodies is constructed from components of the variable
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P. M. Colman
Fig. 3. Drawings of the N2 neuraminidase subunit structure oriented as in Fig. 2. Colour coding shows sites of amino acid sequence variation among characterized isolates of N2 subtype^ in the periods 1957-68 (red). 1968-72 (yellow), 1972-75 (green) and 1975-79 (blue). The maximum dimensions of the subunit are approximately 50 A (left-hand view) and approximately 60 A (right-hand view) and the footprint of an antibody is of the order of the size of a circle of diameter 25-30 A.
Fig. 4. Some examples of amino acid substitutions observed in monoclonal variants of influenza haemagglutinin and neuraminidase plotted on a French-Robson'" distribution of the 20 amino acids. (Adapted from Colman and
domains of both heavy and light chains (Fig. 5). It follows then that the interface between the VH and V^ domains is itself a determinant of antibody specificity since juxtaposition of the two halves of the binding site is determined by that interface.'^ P sheet structures are very common in proteins as are higher order structures composed of assemblies of P sheets. With very few exceptions, the association of two p sheets with each other can be described as belonging to one of two categories. Either the strand directions in the two sheets are 'aligned' or they are 'orthogonal'. In immunoglobulins, the basic domain structure exemplifies the 'aligned' class while the association of constant
Structural basis of antigenic variation
213
Fig. 5. Ca skeleton models of neuraminidase subunit (orange) and variable domains of heavy (blue) and light chain (green) of NC41 antibody. Interacting surfaces are shown in yellow and blue and the antigen and antibody have been separated by 8 A for clarity of presentation.
domains as seen for example in Fab fragments is typical of the 'orthogonal' class.'^ Given the preponderance of these two structural classes in proteins, it is remarkable that the interface between the variable domains of an immunoglobulin should conform to neither. In that case the strand directions are intermediate between aligned and orthogonal, and, furthermore, the interaction is dominated by the edge strands of the two sheets and not the central strands as is more common. It has been suggested that this interface might be required to perform as a semiflexible adapter^ ^ during the process of engagement of antigen by antibody and two reports now support this hypothesis. Studies with an anti-lysozyme antibody with and without bound antigen have shown that a small rearrangement at the interface of the variable domains distinguishes the unliganded
from the liganded structure.'* A similar result has been reported for an anti-single-stranded DNA antibody.'^
References 1. Krug, R. M. 1989 (ed.). The Influenza Viruses. Plenum, New York. 2. Varghese, J. N., Laver, W. G. and Colman, P. M. 1983. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature 303; 35-40. 3. Varghese, j . N. and Colman, P. M. 1991. Three-dimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution./ Mol. Biol. 221: 473-486. 4. Tulip, W. R., Varghese, J. N., Baker, A. T. et al. 1991. Refmed atomic structures of N9 subtype influenza virus neuraminidase and escape mutants/ Mol. Biol. 221: 487-497.
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P. M. Colman
5. Colman, P. M. 1989. The Influenza Viruses, pp. 175-218. Plenum, New York. 6. Webster, R. G.. Hinshaw, V. S. and Laver, W. G. 1982. Selection and analysis of antigenic variants of the neuraminidase of N2 influenza virus with monoclonal antibodies. ^'iVo/o^ 117; 93-104. 7. Webster. R. G.. Air, G. M., Metzger, D. W. et al. 1987. Antigenic structure and variation in an influenza virus N9 neuraminidase./ Virol. 61:2910. 8. Knossow, M., Daniels. R. S., Douglas, A. R., Skehel J. J. and Wiley, D. C. 1984. Threedimensional structure of an antigenic mutant of the influenza virus haemagglutinin. Nature 311:678-680. 9. Varghese, J. N.. Webster, R. G.. Uver. W. G. and Colman, P. M. 1988. Structure of an escape mutant of glycoprotein N2 neuraminidase of influenza virus A/Tokyo/3/67 at 3 A resolution./ Mol. Biol. 200: 201-203. 10. Colman, P. M., Uver, W. G., Varghese. J. N. et al. 1987. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. Nature 526: 358-363. 11. Colman. P. M., Tulip, W. R., Varghese, J. N. et al. 1989. Three-dimensional structures of influenza virus neuraminidase-antibody complexes. Phil. Trans. R. Soc. Lond. (Biol) 323: 511-518. 12. Tulip. W. R., Varghese. J. N.. Webster, R. G.. Air. G. M., Uver. W. G. and Colman. P. M,
13.
14.
15.
16.
17.
18. 19.
1989. Crystal structures of neuraminidaseantibody complexes. Cold Spring Harb. Symp. Quant. Biol. 54: 257-263. Davies, D. R. and Padlan, E. A.. 1990 antibody-antigen complexes. Annu. R(T. Biochem. 59: 439-473. Bentley, G. A., Boulot. G., Riottot. M. M. and Poljak, R. J. 1990. Three dimensional structure of an idiotope-antiidiotope complex. Nature 348: 254-257. Colman. P. M. 1988 Structure of antibodyantigen complexes: Implications for immune recognition. Adv. Immunol. 43: 99-132. Bhat, T. N., Bentley, G. A., Fischmann. T. O., Boulot, G., Poljak, R. J. 1990. Small rearrangements in structures of Fy and Fab fragments of antibody D1.3 on antigen binding. Nature, 347: 483-485. Herron, J. N.. He, X. M., Ballard, D. W. et at. 1992. An autoantibody to single-stranded DNA; comparison of tbe three dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins 11: 159-175. French, S. and Rohson. B. 1983. What is a conservative substitution? / Mol. Evol. 19: 171-175. Colman, P. M. and Ward, C. W. 1985 Structure and diversity of influenza virus neuraminidase. Curr. Top. Microhiot. Immunol. 114: 117255.
Structural basis of antigenic variation: Studies of influenza virus neuraminidase p. M. COLMAN CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia Summary Recent studies of the structural basis of antigenic variation in influenza virus neuraminidase and of the structure of neuraminidase-antibody complexes are summarized.
Introduction The immune system has a remarkable capacity to generate a large number of binding specificities among the family of proteins which act as receptors for antigen. The terms 'binding' and 'specificity' are commonly used in biology to describe the characteristics of molecular interactions, but the structural principles which determine the response of two molecules to each other are both complex and subtle. Studies of antibody-anti gen interactions in solution have illustrated both of these characteristics. The complexity of the phenomenon is highlighted by the capacity of antibodies to cross-react with proteins unrelated to the immunizing antigen, and its subtlety is underscored by the failure of monoclonal antibodies to bind to single site variants of antigen in which the amino acid substitution is conservative. Studies by X-ray crystallography of the three-dimensional structure of influenza virus neuraminidase and of its complex with monoclonal antibodies have now revealed some of the structural principles which govern the formation of complexes. This paper summarizes recent work.
Antigenic variation in influenza viruses The two envelope-associated antigens of type A influenza virus, neuraminidase and haemagglutinin, display structural variation from strain to strain, which has previously been reviewed by Krug.' Antigenic subtypes of both the neuraminidase and haemagglutinin are defined by the capacity ot antisera to cross-react with other members of the group, and within such groups the amino acid sequences of the antigens differ by about 10-20%. Sequence differences between subtypes are of the order of 50%. Nine subtypes of neuraminidase have been identified and the three-dimensional structures of two of them, N2~'^ and N9'* have been determined by X-ray crystallography. Within the soluble 'head' portion of the molecule which can be cut from the viral membrane with enzyme, the N2 and N9 sequences are 48% identical. In two places in the sequence N9 has insertions with respect to N2 and in two other places it has single residue deletions. The three-dimensional structures of the two antigens are very similar at the level of the polypeptide fold, each structure displaying a
Correspondence: P. M. Colman, CSIRO Division of Biomolecular Engineering. 343 Royal Parade, Parkville, Vic. 3052, Australia. Presented at the 4th Frank and Bobbie Fenner Conference in Medical Research on Getietk Variation and Selection in Microbiology and Immunology, held on 6-8 November 1991 at the John Curtin School of Medical Research, Canberra, Australia. Accepted for publication 6 November 1991.
210
P. M Colman
Fig. 1. Schematic drawing of the neuraminidase monomer. The N-terminal residue of the enzyme solubilized neuraminidase is near residue 80 and is on the under-side in this view, presumably the membrane-proximal side of the molecule. The chain folds in six topologically identical fourstranded P-sheets. (Reproduced from Varghese and Colman.") six-bladed p sheet propeller motif (Fig. 1). The region of the structure in which the amino acid sequence is most variable, both within and between subtypes, is the segment linking p sheets 4 and 5, residues 320-350. There also the three-dimensional structures of N2 and N9 are least similar.'* The failure of antisera to cross-react between N2 and N9 structures can be understood in terms of what is now known about the size of a binding site for antibody on an antigen. Approximately 700 A^ of surface area is usually abstracted from solvent when an antibody-antigen complex forms. Figure 2 shows two orthogonal views of the N9 structure,** colour coded to indicate regions of amino acid sequence identity with N2. The largest such contiguous region on the surface of the molecule is of the order of 500 A^ seen in the upper right ofthe left-hand panel. This is the enzyme active site and it is probably too small to bind to an antibody. In contrast, field strain variation within subtypes results in large areas of exposed surface being unmodified from year to year, and these are presumably the basis of crossreaction by antisera. For example, among the
known sequences of N2 strain.^ the neuraminidase is altered at some 27 residues between the years 1957 and 1968 and at a further 16 positions between 1968 and 1972 (Fig. 3). Large regions of the surface remain conserved during these periods and they are presumably the targets of cross-reacting antibodies. The foregoing discussion has all related to field strain variation ofthe antigens. There are now many examples of laboratory variants, the most interesting of which have been selected with monoclonal antibodies.'''' Typically these variants differ from wild type by a single amino acid substitution. That substitution suffices effectively to abolish the binding of the monoclonal antibody to the wild type antigen against which it is raised. The types of substitution seen in these experiments include most ofthe amino acid exchanges possible by single base substitution (Fig. 4). A number of three-dimensional structures of monoclonal variants have now been studied for the haemagglutinin^ and neuraminidase,^'^ and in all cases to date they show only local structural perturbation at, or immediately adjacent to, the site of the amino acid substitution.
Neuratninidase-antibody complexes Two monoclonal antibodies to influenza vims N9 neuraminidase, NC41 and NCIO, have been studied by crystallography as complexes with the antigen.^ '^ Forthe NC41 complex the central findings are as follows. Five linearly discontinuous surface loops of the neuraminidase contact the antibody through five of its six complementarity-determining regions (CDR). Nineteen amino acids on the antigen are in contact with 17 on the antibody. The surface areas buried by antigen and antibody as a consequence of complex formation are each 880 A (Fig. 5). There are 12 hydrogen bonds and three ion pairs in the interface. These observations are similar to those of others working on the lysozyme-antibody complexes (reviewed by Davies and Padlan'^). In the case ofthe neuraminidase complex, the interaction is somewhat more extensive.
Structural basis of antigenic variation
211
Fig. 2. Space-filling shadowed models of N9 neurarainidase showing in different colours those residues identical in N9 and N2 (white) and residues of different amino acid type (red). Left. The top surface ofthe neuraminidase is shown with the four-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 four-fold axis at the middle rear. Antisera to one subtype could not cross-react with the other because there is no common spatial epitope ofthe required size (approximately 800 A^). (Reproduced from Tulip etal.'*)
The NCIO structure is not yet complete, but it is quite clear that it binds an epitope overlapping that of NC41. The detailed analysis of that structure will demonstrate how two different antibodies bind the same structures on an antigen. This phenomenon has a parallel in anti-idiotypy, where an antibody raised to an immunizing antigen can itself be used to raise an antibody response directed at its CDR. The resulting anti-idiotypic antibody and the initial antigen do not necessarily share obvious structural characteristics, although they both bind the primary antibody. One antibodyanti-idiotypic antibody complex structure has
been studied by X-ray crystallography,^** demonstrating that the antigen, lysozyme in that case, and the anti-idiotypic antibody have little in common. The anti-neuraminidase antibodies have not yet been studied in their unliganded form to permit a comparison of the bound and free structures.
Antibody structure and binding properties The binding site for antigen on antibodies is constructed from components of the variable
212
P. M. Colman
Fig. 3. Drawings of the N2 neuraminidase subunit structure oriented as in Fig. 2. Colour coding shows sites of amino acid sequence variation among characterized isolates of N2 subtype^ in the periods 1957-68 (red). 1968-72 (yellow), 1972-75 (green) and 1975-79 (blue). The maximum dimensions of the subunit are approximately 50 A (left-hand view) and approximately 60 A (right-hand view) and the footprint of an antibody is of the order of the size of a circle of diameter 25-30 A.
Fig. 4. Some examples of amino acid substitutions observed in monoclonal variants of influenza haemagglutinin and neuraminidase plotted on a French-Robson'" distribution of the 20 amino acids. (Adapted from Colman and
domains of both heavy and light chains (Fig. 5). It follows then that the interface between the VH and V^ domains is itself a determinant of antibody specificity since juxtaposition of the two halves of the binding site is determined by that interface.'^ P sheet structures are very common in proteins as are higher order structures composed of assemblies of P sheets. With very few exceptions, the association of two p sheets with each other can be described as belonging to one of two categories. Either the strand directions in the two sheets are 'aligned' or they are 'orthogonal'. In immunoglobulins, the basic domain structure exemplifies the 'aligned' class while the association of constant
Structural basis of antigenic variation
213
Fig. 5. Ca skeleton models of neuraminidase subunit (orange) and variable domains of heavy (blue) and light chain (green) of NC41 antibody. Interacting surfaces are shown in yellow and blue and the antigen and antibody have been separated by 8 A for clarity of presentation.
domains as seen for example in Fab fragments is typical of the 'orthogonal' class.'^ Given the preponderance of these two structural classes in proteins, it is remarkable that the interface between the variable domains of an immunoglobulin should conform to neither. In that case the strand directions are intermediate between aligned and orthogonal, and, furthermore, the interaction is dominated by the edge strands of the two sheets and not the central strands as is more common. It has been suggested that this interface might be required to perform as a semiflexible adapter^ ^ during the process of engagement of antigen by antibody and two reports now support this hypothesis. Studies with an anti-lysozyme antibody with and without bound antigen have shown that a small rearrangement at the interface of the variable domains distinguishes the unliganded
from the liganded structure.'* A similar result has been reported for an anti-single-stranded DNA antibody.'^
References 1. Krug, R. M. 1989 (ed.). The Influenza Viruses. Plenum, New York. 2. Varghese, J. N., Laver, W. G. and Colman, P. M. 1983. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature 303; 35-40. 3. Varghese, j . N. and Colman, P. M. 1991. Three-dimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution./ Mol. Biol. 221: 473-486. 4. Tulip, W. R., Varghese, J. N., Baker, A. T. et al. 1991. Refmed atomic structures of N9 subtype influenza virus neuraminidase and escape mutants/ Mol. Biol. 221: 487-497.
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P. M. Colman
5. Colman, P. M. 1989. The Influenza Viruses, pp. 175-218. Plenum, New York. 6. Webster, R. G.. Hinshaw, V. S. and Laver, W. G. 1982. Selection and analysis of antigenic variants of the neuraminidase of N2 influenza virus with monoclonal antibodies. ^'iVo/o^ 117; 93-104. 7. Webster. R. G.. Air, G. M., Metzger, D. W. et al. 1987. Antigenic structure and variation in an influenza virus N9 neuraminidase./ Virol. 61:2910. 8. Knossow, M., Daniels. R. S., Douglas, A. R., Skehel J. J. and Wiley, D. C. 1984. Threedimensional structure of an antigenic mutant of the influenza virus haemagglutinin. Nature 311:678-680. 9. Varghese, J. N.. Webster, R. G.. Uver. W. G. and Colman, P. M. 1988. Structure of an escape mutant of glycoprotein N2 neuraminidase of influenza virus A/Tokyo/3/67 at 3 A resolution./ Mol. Biol. 200: 201-203. 10. Colman, P. M., Uver, W. G., Varghese. J. N. et al. 1987. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. Nature 526: 358-363. 11. Colman. P. M., Tulip, W. R., Varghese, J. N. et al. 1989. Three-dimensional structures of influenza virus neuraminidase-antibody complexes. Phil. Trans. R. Soc. Lond. (Biol) 323: 511-518. 12. Tulip. W. R., Varghese. J. N.. Webster, R. G.. Air. G. M., Uver. W. G. and Colman. P. M,
13.
14.
15.
16.
17.
18. 19.
1989. Crystal structures of neuraminidaseantibody complexes. Cold Spring Harb. Symp. Quant. Biol. 54: 257-263. Davies, D. R. and Padlan, E. A.. 1990 antibody-antigen complexes. Annu. R(T. Biochem. 59: 439-473. Bentley, G. A., Boulot. G., Riottot. M. M. and Poljak, R. J. 1990. Three dimensional structure of an idiotope-antiidiotope complex. Nature 348: 254-257. Colman. P. M. 1988 Structure of antibodyantigen complexes: Implications for immune recognition. Adv. Immunol. 43: 99-132. Bhat, T. N., Bentley, G. A., Fischmann. T. O., Boulot, G., Poljak, R. J. 1990. Small rearrangements in structures of Fy and Fab fragments of antibody D1.3 on antigen binding. Nature, 347: 483-485. Herron, J. N.. He, X. M., Ballard, D. W. et at. 1992. An autoantibody to single-stranded DNA; comparison of tbe three dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins 11: 159-175. French, S. and Rohson. B. 1983. What is a conservative substitution? / Mol. Evol. 19: 171-175. Colman, P. M. and Ward, C. W. 1985 Structure and diversity of influenza virus neuraminidase. Curr. Top. Microhiot. Immunol. 114: 117255.
