- Email: [email protected]
Comparison of CH1 Domains in Different Classes of Murine Antibodies
J. Mol. Biol. (1996) 263, 385–389
COMMUNICATION
Comparison of CH 1 Domains in Different Classes of Murine Antibodies Steven Sheriff1*, Philip D. Jeffrey1 and Ju¨rgen Bajorath2 1
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000 Princeton, NJ 08543-4000 USA 2
Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue Seattle, WA 98121, USA
The CH 1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG CH 1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG CH 1 domain structures, the IgA CH 1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG CH 1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for CH 1 domains has been revised to be consistent with the three-dimensional alignments. 7 1996 Academic Press Limited
*Corresponding author
Keywords: immunoglobulin structure; antibody classes; structure comparison; structure-oriented sequence comparison; Kabat numbering
The three-dimensional structures of Fabs from all the murine immunoglobulin (IgG) classes (IgG1, IgG2a, IgG2b and IgG3) and the murine IgA class have been determined. This database allows the comparison of the structures of murine Ig heavy chain constant domains. The five CH 1 domains have been superimposed and the superposition has been related to the alignment of sequences in the Kabat et al. (1991) database similar to that of Chothia et al. (1989) for complementarity determining loops. The structures of the IgG CH 1 domains are very similar, but not invariant. The best spatial superpositions of the IgG CH 1 domains are not consistent with the conventional alignment and numbering scheme used for these domains (Kabat et al., 1991). Moreover, comparison of the IgG CH 1 domains with the IgA CH 1 domain reveal significant conformational differences and the presence of insertions/deletions, which is not properly reflected by the Kabat alignment and numbering scheme. Therefore, structure-oriented revisions of sequence alignment and numbering for parts of the CH 1 domains are proposed. Thus the Present address: P. D. Jeffrey, Box 576, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Abbreviations used: Fab, antigen-binding fragment produced by papain; CH 1, first constant domain of the heavy chain; VH , variable domain of the heavy chain; r.m.s.d. root-mean-square deviation. 0022–2836/96/430385–05 $25.00/0
observed structure differences in the CH 1 domains demonstrate that constant domains must be carefully selected in attempting to build three-dimensional models of other members of the immunoglobulin superfamily. Optimal three-dimensional alignments of Ca positions were generated using program ALIGN written by G. H. Cohen (Satow et al., 1986). ALIGN discerns optimal positioning based on pairwise comparisons and deletes atom pairs from the comparison if they exceed three times the r.m.s. deviation for the superposition of all pairs compared. The positions of insertions and deletions in IgA relative to the IgG CH 1 domains were assigned on the basis of the best calculated pairwise Ca superpositions. Structures for the classes of antibodies were selected on the basis of the highest sequence identity with the class sequences (Kabat et al., 1991), the highest resolution and the lowest crystallographic R-value for the class. The antibodies chosen from the Protein Data Bank (Bernstein et al., 1977) for the comparison are listed in Table 1. As a reference for conformational variation within the same structure, 17/9 (1HIL) has two Fabs in the asymmetric unit, so the CH 1 of chain B was compared with chain D (Table 1). Figure 1 shows the proposed sequence alignment based on three-dimensional superposition of each of the CH 1 domains on that of the IgG2a CH 1 domain (17/9; 1HIL chain B). Nineteen of the 20 Ca 7 1996 Academic Press Limited
386
Comparison of Murine CH 1 Domains
Table 1. Antibodies compared Class
Antibody
PDB
˚) Res. (A
R
˚) r.m.s.d. (A
Reference
IgG1 IgG2a IgG2b IgG3 IgA
D44.1 17/9 BV04-01 BR96 J539
1MLB 1HIL 1NBV 1CLZ 2FBJ
2.1 2.0 2.0 2.8 1.95
0.181 0.195 0.246 0.197 0.194
0.38 (0.46) 0.12 (0.18) 0.40 (0.69) 0.49 (0.70) 0.68 (1.06)
Braden et al. (1994) Rini et al. (1992) Herron et al. (1991) Jeffrey et al. (1995) Suh et al. (1986)
Comment Chain B
The r.m.s. deviations were obtained for the Ca positions for 19 identical residues according to Figure 1 (Pro199 was excluded, because it is cis in the IgGs and trans in the IgA). All CH 1 domains were superimposed on the CH 1 domain of chain B of 17/9. In parentheses, r.m.s.d. values are given for Ca positions for the 73 spatially corresponding residues based on the superposition of the 19 identical residues. Residues excluded from the comparison were 115, 127 to 136, 163 to 168, 200 to 204 and 214 to 217 (Figure 1). The entry 1HIL has two Fabs in the asymmetric unit with heavy chains labeled B and D. The r.m.s.d. comparison for this entry is between chains B and D.
Figure 1. Sequence alignment of murine CH 1 domains based on structural alignment. Residues that are identical across the five classes are highlighted by a gray background. Residues that are identical across the four IgG classes are denoted by boxes. Gaps were included to conform as closely as possible to Kabat et al. (1991) numbering.
positions that are identical in the five sequences (Figure 1) were used for superposition. All r.m.s.d. values reported are based on this superposition. Position H199 in the proposed sequence numbering
(Figure 1) is Pro in all cases, but it is cis in the IgGs and trans in the IgA (see below). Cysteine residues 142 and 208 form the intra-chain Ig disulfide. Proline 149 is cis in all of these structures. An additional 36 residues are identical across all of the IgG classes. Cysteine 128 in IgG2a, 2b and 3 form the inter-domain disulfide with the light chain. Proline 151 is cis in IgG1, 2a and 3. Figure 2 shows the superposition of the compared CH 1 domains. The different IgG CH 1 domains superimpose well overall with the exception of two segments, 127 to 135 and 163 to 168, where coordinates are either not modeled in some structures or B-factors are very high. In the region 163 to 168 the IgG1, IgG2a and IgG3 CH 1 domains show good agreement amongst them˚ ). In the IgG2b CH 1 selves (r.m.s.d. 0.6 to 0.7 A domain structure, however, this region deviates substantially from all of the other IgG CH 1 domains ˚ ). In murine IgG3 antibodies position (r.m.s.d. 07 A 114 (last residue in VH ) is absent. In addition to differences in the VH-CH 1 domain-domain
Figure 2. Stereo view of the superposition of the CH 1 of murine IgG2a with the CH 1 of murine IgG1, IgG2b, IgG3, and IgA. Color code: IgG1 (magenta), IgG2a (red), IgG2b (cyan), IgG3 (orange), and IgA (blue). Numbering according to Figure 1. The Figure was produced with the program MOLSCRIPT (Kraulis, 1991) using the ‘‘turn’’ command.
Comparison of Murine CH 1 Domains
387
(a)
(b)
(c)
(d)
Figure 3. Stereo views of regions with insertions/deletions. (a) Residues 150 to 156; (b) residues 179 to 184; (c) residues 197 to 207 with side-chains beyond Cb omitted for clarity; (d) residues 213 to 219. Color code: IgG1 (magenta), IgG2a (red), IgG2b (cyan), IgG3 (orange), and IgA (blue). Numbering according to Figure 1 in black, with IgA numbering as necessary in red. The Figure was produced with the program MOLSCRIPT (Kraulis, 1991).
388
Comparison of Murine CH 1 Domains
Table 2. Best fit of IgA and IgG1 CH 1 domain segments with insertions and deletions A. Residues 151 to 155 Residue number Proposed IgG IgA 151 152 153 154 155
151 152 153 154
B. Residues 180 to 184 180 182 180 183 183 184 184
Ca position ˚) difference (A
151 152 153 154 155
2.1–2.7 1.6–2.0 1.7–1.8 0.9–1.2
180 181 182 183
1.8–2.4 0.3–0.6 0.4–0.8
C. Residues 197 to 207 Residue number Proposed IgG IgG1 197 198 199 200 202 203 204 205 206 207
198 199 200
199 200 202
202 203 204 205 206
203 204 205 206 207
D. Residues 215 to 219 Residue number Proposed IgG IgA 215 216 217 218 219
215 216 217 218 219
215 216 217 218
IgA 197 198 199 200 202 203 204 205 206
Ca position ˚) difference (A IgG1 IgA 0.8–1.6 0.6–1.3 0.7–1.7
1.4–3.0 1.2–2.3 2.8–4.3
0.8–1.9 0.7–1.3 0.7–1.3 0.5–0.7 0.3–0.5
3.3–5.4 3.1–3.5 0.6–1.1 0.6–0.9
Ca position ˚) difference (A 1.3–2.7 4.0–4.8 1.8–2.7 0.8–1.9
Residue numbering in columns labeled IgG, IgG1 and IgA is that of Kabat et al. (1991). The IgG column refers to the Kabat numbering for IgG1, IgG2a, IgG2b and IgG3 in A, B and D. In C, the IgG column refers to the Kabat numbering for IgG2a, IgG2b and IgG3.
orientation, the absence of this residue contributes to differences in the backbone trace at the beginning of CH 1 domains. In IgG1 CH 1 domains the segment 197 to 207 (199 to 207 Kabat numbering) superimposes with r.m.s.d. of 0.7 to ˚ on the other IgGs (198 to 206 Kabat 1.3 A numbering; Table 2C and Figure 3c). Therefore, neither the structural similarity nor even the sequence similarity is reflected in the Kabat numbering scheme in this region (Figure 3(c) and Table 2C). On the basis of the superpositions, alternative sequence numbers are proposed (Figure 1). Excluding residues 115, 127 to 135 and 163 to 168, the r.m.s.d. values for the best superpositions ˚ for of the four IgG classes range from 0.5 to 0.8 A a 81 C positions. When the IgA CH 1 is compared with the IgG CH 1 domains, four segments show insertions/deletions (151 to 154, 180 to 183, 198 to 204, 214 to 218; Figures 2 and 3). Table 2 summarizes the positions of insertions and deletions in the segments as suggested by the alignments of Ca positions. For
example, Kabat numbering (Table 2A) suggests that position 155 of IgA is an insertion residue relative to the IgG classes, but three-dimensional superposition suggests that IgA position 152 is the insertion residue in this region. The backbone conformation of the IgA CH 1 domain differs significantly from the IgG structures in the segment 199 to 205 (r.m.s.d. ˚ to 4.2 A ˚ ). Analysis of for three Ca positions of 3.1 A these differences show that the IgA CH 1 domain has a trans proline residue at position 199 in contrast to a cis proline residue at 199, which is conserved in the murine IgG CH 1 domains. Furthermore, in the IgA CH 1 domain a glutamate residue at position 200 is an insertion and a glycine residue at position 202 adopts torsion angles not usually allowed for other residue types, whereas in the IgG CH 1 domains position 203 is a serine residue with allowed torsion angles. The analysis suggests that these conformational differences are a consequence of classspecific structural constraints. The structural differences revealed by comparison of the murine CH 1 domains suggests the extension of the analysis to other Ig constant domains.
Acknowledgements We thank the editor and referees for helpful comments.
References Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr, Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977). The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542. Braden, B. C., Souchon, H., Eisele´, J.-L., Bentley, G. A., Bhat, T. N., Navaza, J. & Poljak, R. J. (1994). Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal antilysozyme antibody, D44.1. J. Mol. Biol. 243, 767– 781. Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., Colman, P. M., Spinelli, S., Alzari, P. M. & Poljak, R. J. (1989). The conformations of immunoglobulin hypervariable regions. Nature, 342, 877–883. Herron, J. N., He, X. M., Ballard, D. W., Blier, P. R., Pace, P. E., Bothwell, A. L. M., Voss, E. W., Jr & Edmundson, A. B. (1991). An autoantibody to single-stranded DNA: comparison of the threedimensional structures of the unliganded Fab and a deoxynucleotide–Fab complex. Proteins: Struct. Funct. Genet. 11, 159–175. Jeffrey, P. D., Bajorath, J., Chang, C. Y., Yelton, D., Hellstro¨m, I., Hellstro¨m, K. E. & Sheriff, S. (1995). The X-ray structure of an anti-tumour antibody in complex with antigen. Nature Struct. Biol. 2, 466–471. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., National Institutes of Health, Bethesda, MD.
Comparison of Murine CH 1 Domains
Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946–950. Rini, J. M., Schulze-Gahmen, U. & Wilson, I. A. (1992). Structural evidence for induced fit as a mechanism for antibody-antigen recognition. Science, 255, 959– 965. Satow, Y., Cohen, G. H., Padlan, E. A. & Davies, D. R.
389 (1986). Phosphocholine binding immunoglobulin ˚. Fab McPC603. An X-ray diffraction study at 2.7 A J. Mol. Biol. 190, 593–604. Suh, S. W., Bhat, T. N., Navia, M. A., Cohen, G. H., Rao, D. N., Rudikoff, S. & Davies, D. R. (1986). The galactan-binding immunoglobulin Fab J539: an X-ray ˚ resolution. Proteins: Struct. diffraction study at 2.6-A Funct. Genet, 1, 74–80.
Edited by I. A. Wilson (Received 4 June 1996; received in revised form 26 July 1996; accepted 29 July 1996)
COMMUNICATION
Comparison of CH 1 Domains in Different Classes of Murine Antibodies Steven Sheriff1*, Philip D. Jeffrey1 and Ju¨rgen Bajorath2 1
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000 Princeton, NJ 08543-4000 USA 2
Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue Seattle, WA 98121, USA
The CH 1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG CH 1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG CH 1 domain structures, the IgA CH 1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG CH 1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for CH 1 domains has been revised to be consistent with the three-dimensional alignments. 7 1996 Academic Press Limited
*Corresponding author
Keywords: immunoglobulin structure; antibody classes; structure comparison; structure-oriented sequence comparison; Kabat numbering
The three-dimensional structures of Fabs from all the murine immunoglobulin (IgG) classes (IgG1, IgG2a, IgG2b and IgG3) and the murine IgA class have been determined. This database allows the comparison of the structures of murine Ig heavy chain constant domains. The five CH 1 domains have been superimposed and the superposition has been related to the alignment of sequences in the Kabat et al. (1991) database similar to that of Chothia et al. (1989) for complementarity determining loops. The structures of the IgG CH 1 domains are very similar, but not invariant. The best spatial superpositions of the IgG CH 1 domains are not consistent with the conventional alignment and numbering scheme used for these domains (Kabat et al., 1991). Moreover, comparison of the IgG CH 1 domains with the IgA CH 1 domain reveal significant conformational differences and the presence of insertions/deletions, which is not properly reflected by the Kabat alignment and numbering scheme. Therefore, structure-oriented revisions of sequence alignment and numbering for parts of the CH 1 domains are proposed. Thus the Present address: P. D. Jeffrey, Box 576, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Abbreviations used: Fab, antigen-binding fragment produced by papain; CH 1, first constant domain of the heavy chain; VH , variable domain of the heavy chain; r.m.s.d. root-mean-square deviation. 0022–2836/96/430385–05 $25.00/0
observed structure differences in the CH 1 domains demonstrate that constant domains must be carefully selected in attempting to build three-dimensional models of other members of the immunoglobulin superfamily. Optimal three-dimensional alignments of Ca positions were generated using program ALIGN written by G. H. Cohen (Satow et al., 1986). ALIGN discerns optimal positioning based on pairwise comparisons and deletes atom pairs from the comparison if they exceed three times the r.m.s. deviation for the superposition of all pairs compared. The positions of insertions and deletions in IgA relative to the IgG CH 1 domains were assigned on the basis of the best calculated pairwise Ca superpositions. Structures for the classes of antibodies were selected on the basis of the highest sequence identity with the class sequences (Kabat et al., 1991), the highest resolution and the lowest crystallographic R-value for the class. The antibodies chosen from the Protein Data Bank (Bernstein et al., 1977) for the comparison are listed in Table 1. As a reference for conformational variation within the same structure, 17/9 (1HIL) has two Fabs in the asymmetric unit, so the CH 1 of chain B was compared with chain D (Table 1). Figure 1 shows the proposed sequence alignment based on three-dimensional superposition of each of the CH 1 domains on that of the IgG2a CH 1 domain (17/9; 1HIL chain B). Nineteen of the 20 Ca 7 1996 Academic Press Limited
386
Comparison of Murine CH 1 Domains
Table 1. Antibodies compared Class
Antibody
PDB
˚) Res. (A
R
˚) r.m.s.d. (A
Reference
IgG1 IgG2a IgG2b IgG3 IgA
D44.1 17/9 BV04-01 BR96 J539
1MLB 1HIL 1NBV 1CLZ 2FBJ
2.1 2.0 2.0 2.8 1.95
0.181 0.195 0.246 0.197 0.194
0.38 (0.46) 0.12 (0.18) 0.40 (0.69) 0.49 (0.70) 0.68 (1.06)
Braden et al. (1994) Rini et al. (1992) Herron et al. (1991) Jeffrey et al. (1995) Suh et al. (1986)
Comment Chain B
The r.m.s. deviations were obtained for the Ca positions for 19 identical residues according to Figure 1 (Pro199 was excluded, because it is cis in the IgGs and trans in the IgA). All CH 1 domains were superimposed on the CH 1 domain of chain B of 17/9. In parentheses, r.m.s.d. values are given for Ca positions for the 73 spatially corresponding residues based on the superposition of the 19 identical residues. Residues excluded from the comparison were 115, 127 to 136, 163 to 168, 200 to 204 and 214 to 217 (Figure 1). The entry 1HIL has two Fabs in the asymmetric unit with heavy chains labeled B and D. The r.m.s.d. comparison for this entry is between chains B and D.
Figure 1. Sequence alignment of murine CH 1 domains based on structural alignment. Residues that are identical across the five classes are highlighted by a gray background. Residues that are identical across the four IgG classes are denoted by boxes. Gaps were included to conform as closely as possible to Kabat et al. (1991) numbering.
positions that are identical in the five sequences (Figure 1) were used for superposition. All r.m.s.d. values reported are based on this superposition. Position H199 in the proposed sequence numbering
(Figure 1) is Pro in all cases, but it is cis in the IgGs and trans in the IgA (see below). Cysteine residues 142 and 208 form the intra-chain Ig disulfide. Proline 149 is cis in all of these structures. An additional 36 residues are identical across all of the IgG classes. Cysteine 128 in IgG2a, 2b and 3 form the inter-domain disulfide with the light chain. Proline 151 is cis in IgG1, 2a and 3. Figure 2 shows the superposition of the compared CH 1 domains. The different IgG CH 1 domains superimpose well overall with the exception of two segments, 127 to 135 and 163 to 168, where coordinates are either not modeled in some structures or B-factors are very high. In the region 163 to 168 the IgG1, IgG2a and IgG3 CH 1 domains show good agreement amongst them˚ ). In the IgG2b CH 1 selves (r.m.s.d. 0.6 to 0.7 A domain structure, however, this region deviates substantially from all of the other IgG CH 1 domains ˚ ). In murine IgG3 antibodies position (r.m.s.d. 07 A 114 (last residue in VH ) is absent. In addition to differences in the VH-CH 1 domain-domain
Figure 2. Stereo view of the superposition of the CH 1 of murine IgG2a with the CH 1 of murine IgG1, IgG2b, IgG3, and IgA. Color code: IgG1 (magenta), IgG2a (red), IgG2b (cyan), IgG3 (orange), and IgA (blue). Numbering according to Figure 1. The Figure was produced with the program MOLSCRIPT (Kraulis, 1991) using the ‘‘turn’’ command.
Comparison of Murine CH 1 Domains
387
(a)
(b)
(c)
(d)
Figure 3. Stereo views of regions with insertions/deletions. (a) Residues 150 to 156; (b) residues 179 to 184; (c) residues 197 to 207 with side-chains beyond Cb omitted for clarity; (d) residues 213 to 219. Color code: IgG1 (magenta), IgG2a (red), IgG2b (cyan), IgG3 (orange), and IgA (blue). Numbering according to Figure 1 in black, with IgA numbering as necessary in red. The Figure was produced with the program MOLSCRIPT (Kraulis, 1991).
388
Comparison of Murine CH 1 Domains
Table 2. Best fit of IgA and IgG1 CH 1 domain segments with insertions and deletions A. Residues 151 to 155 Residue number Proposed IgG IgA 151 152 153 154 155
151 152 153 154
B. Residues 180 to 184 180 182 180 183 183 184 184
Ca position ˚) difference (A
151 152 153 154 155
2.1–2.7 1.6–2.0 1.7–1.8 0.9–1.2
180 181 182 183
1.8–2.4 0.3–0.6 0.4–0.8
C. Residues 197 to 207 Residue number Proposed IgG IgG1 197 198 199 200 202 203 204 205 206 207
198 199 200
199 200 202
202 203 204 205 206
203 204 205 206 207
D. Residues 215 to 219 Residue number Proposed IgG IgA 215 216 217 218 219
215 216 217 218 219
215 216 217 218
IgA 197 198 199 200 202 203 204 205 206
Ca position ˚) difference (A IgG1 IgA 0.8–1.6 0.6–1.3 0.7–1.7
1.4–3.0 1.2–2.3 2.8–4.3
0.8–1.9 0.7–1.3 0.7–1.3 0.5–0.7 0.3–0.5
3.3–5.4 3.1–3.5 0.6–1.1 0.6–0.9
Ca position ˚) difference (A 1.3–2.7 4.0–4.8 1.8–2.7 0.8–1.9
Residue numbering in columns labeled IgG, IgG1 and IgA is that of Kabat et al. (1991). The IgG column refers to the Kabat numbering for IgG1, IgG2a, IgG2b and IgG3 in A, B and D. In C, the IgG column refers to the Kabat numbering for IgG2a, IgG2b and IgG3.
orientation, the absence of this residue contributes to differences in the backbone trace at the beginning of CH 1 domains. In IgG1 CH 1 domains the segment 197 to 207 (199 to 207 Kabat numbering) superimposes with r.m.s.d. of 0.7 to ˚ on the other IgGs (198 to 206 Kabat 1.3 A numbering; Table 2C and Figure 3c). Therefore, neither the structural similarity nor even the sequence similarity is reflected in the Kabat numbering scheme in this region (Figure 3(c) and Table 2C). On the basis of the superpositions, alternative sequence numbers are proposed (Figure 1). Excluding residues 115, 127 to 135 and 163 to 168, the r.m.s.d. values for the best superpositions ˚ for of the four IgG classes range from 0.5 to 0.8 A a 81 C positions. When the IgA CH 1 is compared with the IgG CH 1 domains, four segments show insertions/deletions (151 to 154, 180 to 183, 198 to 204, 214 to 218; Figures 2 and 3). Table 2 summarizes the positions of insertions and deletions in the segments as suggested by the alignments of Ca positions. For
example, Kabat numbering (Table 2A) suggests that position 155 of IgA is an insertion residue relative to the IgG classes, but three-dimensional superposition suggests that IgA position 152 is the insertion residue in this region. The backbone conformation of the IgA CH 1 domain differs significantly from the IgG structures in the segment 199 to 205 (r.m.s.d. ˚ to 4.2 A ˚ ). Analysis of for three Ca positions of 3.1 A these differences show that the IgA CH 1 domain has a trans proline residue at position 199 in contrast to a cis proline residue at 199, which is conserved in the murine IgG CH 1 domains. Furthermore, in the IgA CH 1 domain a glutamate residue at position 200 is an insertion and a glycine residue at position 202 adopts torsion angles not usually allowed for other residue types, whereas in the IgG CH 1 domains position 203 is a serine residue with allowed torsion angles. The analysis suggests that these conformational differences are a consequence of classspecific structural constraints. The structural differences revealed by comparison of the murine CH 1 domains suggests the extension of the analysis to other Ig constant domains.
Acknowledgements We thank the editor and referees for helpful comments.
References Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr, Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977). The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542. Braden, B. C., Souchon, H., Eisele´, J.-L., Bentley, G. A., Bhat, T. N., Navaza, J. & Poljak, R. J. (1994). Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal antilysozyme antibody, D44.1. J. Mol. Biol. 243, 767– 781. Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., Colman, P. M., Spinelli, S., Alzari, P. M. & Poljak, R. J. (1989). The conformations of immunoglobulin hypervariable regions. Nature, 342, 877–883. Herron, J. N., He, X. M., Ballard, D. W., Blier, P. R., Pace, P. E., Bothwell, A. L. M., Voss, E. W., Jr & Edmundson, A. B. (1991). An autoantibody to single-stranded DNA: comparison of the threedimensional structures of the unliganded Fab and a deoxynucleotide–Fab complex. Proteins: Struct. Funct. Genet. 11, 159–175. Jeffrey, P. D., Bajorath, J., Chang, C. Y., Yelton, D., Hellstro¨m, I., Hellstro¨m, K. E. & Sheriff, S. (1995). The X-ray structure of an anti-tumour antibody in complex with antigen. Nature Struct. Biol. 2, 466–471. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., National Institutes of Health, Bethesda, MD.
Comparison of Murine CH 1 Domains
Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946–950. Rini, J. M., Schulze-Gahmen, U. & Wilson, I. A. (1992). Structural evidence for induced fit as a mechanism for antibody-antigen recognition. Science, 255, 959– 965. Satow, Y., Cohen, G. H., Padlan, E. A. & Davies, D. R.
389 (1986). Phosphocholine binding immunoglobulin ˚. Fab McPC603. An X-ray diffraction study at 2.7 A J. Mol. Biol. 190, 593–604. Suh, S. W., Bhat, T. N., Navia, M. A., Cohen, G. H., Rao, D. N., Rudikoff, S. & Davies, D. R. (1986). The galactan-binding immunoglobulin Fab J539: an X-ray ˚ resolution. Proteins: Struct. diffraction study at 2.6-A Funct. Genet, 1, 74–80.
Edited by I. A. Wilson (Received 4 June 1996; received in revised form 26 July 1996; accepted 29 July 1996)