- Email: [email protected]
antigen interactions: Phosphocholine binding to McPC603 and the correlation of three-dimensional structure and sequence data
3-D S T R U C T U R E AND F U N C T I O N OF Ig
271
[14] ROTHSTEIN, T. L. & GEFTER, M. W., Affinity analysis of idiotype-positive and [15] [16] [17]
[13] [19]
idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera. Mol. Immunol., 1983, 20, 161-168. I~UDIKOFF, S., GIUSTI, A. M., COOK, W. D. & SCHARFF, M. D., Single amino acid substitutions altering antigen-binding specificity. Proc. nat. Aead. Sci. ('Wash.), 1982, 79, 1979-1983. SHOENFELD, Y., ISENBERG, D. A., RAUCH, J., MADAIO, M. P., STOLLAR, B. D. & SCHWAnTZ, R. S., Idiotypie cross-reactions of monoclonal human lupus autoantibodies, d. exp. Med., 1983, 158, 718-730. TEILLAUD, J.-L., DESAYMARD, C., GUISTI, A. M., HASELTINE, B., POLLOCK, R. R., YELTON, D. E., ZACK, D. J. & SCHARFF, M. D., Monoclonal antibodies reveal the structural basis of antibody diversity. Science, 1983, 222, 721-726. THEOFILOPOULOS,A. N. & DIXON, F. J., Etiopathogenesis of murine SLE. lmmunol. Rev., 1981, 55, 179-216. TONEGAWX, S., Somatic generation of antibody diversity. Nalure (Lond.), 1983, 302, 575-581.
Acknowledgemenls. The work reported in this paper was supported by grants from the NIH (AI523L, AM32371. A1 10702), the NSF (PCM 836160), the ACS (IM-3LTC), a Cancer Center Grant (3PO CA1330) and the SLE Foundation. B. D. is an established investigator of the American Heart Association and N. C. is supported by a training grant (CA 09173) from the NCI.
ON THE S P E C I F I C I T Y OF A N T I B O D Y / A N T I G E N INTERACTIONS: P H O S P H O C H O L I N E B I N D I N G TO MePC603 AND THE CORRELATION OF T H R E E - D I M E N S I O N A L STRUCTURE AND SEQUENCE DATA
by E. A. Padlan, G. H. Cohen and D. R. Davies
Laboralory o/Molecular Biology, National Institute o[ Arthritis, Diabeies, and Digestive and Kidney Diseases, National Inslilules o/Health, Belhesda, MD, 20205 ( U S A ) Summary. Refined three-dimensional structures of McPC603 Fab and the complex with phosphocholine permit a detailed assessment of the residues crucial to determining the antibody specificity. Correlation with sequence data suggests that the structure of the binding site is highly conserved in immunoglobulins with phosphocholinebinding specificity. There is suggestive evidence that coupling of somatic mutations occurs to preserve antigen-binding specificity. The immune response is characterized b y specificity and diversity. While each antibody appears to be specific for a single antigen, the immune response can
272
6e FORUM D'IMMUNOLOGIE
generate up to 109 different specificities. In order to understand, at the molecular level, the nature of the interaction between antibody and antigen, it is necessary to have a high-resolution three-dimensional picture of the complex. Today it is possible to investigate antibody/antigen interactions directly by the crystallographic analysis of hybridoma products [5, 10]; in the past, structural studies were limited to myeloma proteins which, in some cases, could be shown to complex to certain haptens. Of the four Fab structures that have been determined by X-ray diffraction, only two have been demonstrated to bind hapten in the crystal. They are Fab NEW, which was shown to bind a vitamin K1 derivative [1] and McPC603, which binds to phosphocholine [6, 9]. During the last few years, the McPC603 Fab structure has been refined at 2.7 )k resolution and the complex of McPC603 Fab with phosphocholine has been refined independently at 3.1 )k. In this communication, we make a comparative analysis of the sequences of a number of mouse phosphocholine-binding immunoglobulins based on the refined structure of the phosphocholine-binding site in McPC603. Refinemenl ol the slruclures. The structure of Fab McPC603 has been refined by the application of Hendrickson-Konnert restrained least-squares procedure [3] combined with extensive modelling to a set of 2.7-h resolution intensity data collected photographically by oscillation methods (Satow, Cohen, Padlan and Davies, in preparation). The final R-value is 22.5~o with r. m. s. deviations from ideality of 0.02 A in bond lengths and 3.5 ~ in bond angles. The refined McPC603 Fab structure was used as the starting point for the refinement of the complex with phosphocholine (Padlan, Cohen and Davies, in preparation). The R-value after 12 cycles of HendricksonK o n n e r t refinement is 18.5% with r. m. s. deviations from ideality of 0.014 .~ in bond lengths and 2.8 ~ in bond angles; because of the lower resolution of this refinement, bond distance and angles were held more closely to the ideal values. Resulls. In figure 1 are shown the amino acid residues most closely in contact with phosphocholine in the combining site of McPC603. These residues are T y r 33, Arg 52, Asn 95 and Trp 100a from the heavy chain (the numbering scheme of K a b a t el al. [4] is used here). Light-chain contact residues include Asp 91, T y r 94 and Leu 96 together with main chain atoms of residue 92. The interactions between hapten and these residues involve extensive van der Waals contacts, hydrogen bonds and electrostatic interactions. The hydroxyl group of Tyr 33 is hydrogenbonded to one oxygen of the phosphate. The guanidinium group of Arg 52 is also within hydrogen-bonding distance of phosphate oxygens. The negative charge on the phosphate is to some extent neutralized by the positively charged guanidinium group of Arg 52. The positive charge of the choline moiety is partially neutralized by the side group of Asp 91 of the light chain. In addition to the residues that are in direct contact with the hapten, there are charged side groups that appear to play an ancillary role in the binding of phosphocholine. For example, buried deep in the hapten-binding cavity is Glu 35 of the heavy chain, which can serve to partially neutralize the positive charge of the choline as well as fix the position of T y r 94 of the light chain through a hydrogen bond [7]. Also, the charges on the heavy chain Glu 58 and the lysines at 52b, 54 and 64 could be influential in aligning the hapten as it approaches the cavity [Getzoff and Tainer, personal communication]. Sequence data are available for a number of murine phosphocholine-binding antibodies. In tables I and II are listed the hypervariable region sequences of the heavy and light chains of these proteins [4]. The heavy chain sequences of table I reflect the finding of Gearhart el al. [2] that these proteins are all derived from the
F 32
c~ F 3~
PC
FIG. 1. - -
Stereodrawing oI the combining site o[ .'llcPC603 with phosphocholine (PC) bound.
The lower residues (91-96 a n d F32) are from t h e l i g h t chain; t h e others are from t h e h e a v y chain
3-D STRUCTURE AND FUNCTION OF Ig
275
TABLE I. Heavy-chain hypervariable region sequences of routine phosphocholine-binding immunoglobulins. -
-
CDR 1
MCPC603 TEPCI5 HOPC8 SI07 SIO7.UI HPCMI P,PCM2 HPCM3 IIPCM6 "8PCG8 $63 Y5236 W3207 HPCGI4 MOPCSII HPCGI3 MOPCI67 CBBPC-3 C57BL 293 C57BL 1613 C57BL 2857 C57BL 23169 CBA/N IBSE5 CBA/J 6F9 CBA/J 7C6
CDR 2
31 35 D F Y M E D F Y M E D F Y M E D F Y M E D F Y M E D F Y M E DF YME DFYME DFYME D F Y M E DFYME D F Y M E D F Y M E A F Y M E B F Y M E D F Y M E D F Y M E D F Y M E D F Y H E D F Y M E D F Y M E D F Y Id E D F Y M E D F Y M E D F Y M E
50 52 A S R A S R A S R A S R A S R A S R ASRNK ASRNK ASRNK A S R ASRNK A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R
a N N B N N N
b K K K K K K
N K N B N N N S N N N N N N N N
K K K K K K K K K K K K K K
c53 G N K A N D A N D A N D A N D A N D A N D A N D A N D A F D A N D A N D A N D A N D A N D V Y D A H D A N D A N D A N D A N D A N D A N D A N D A N D
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
T T T T T T T T T T T T T T T T R T T T T T T T T
T T T T T T T T T T T T T T T T T T T T T T T T T
E E Z E E E E E E E E E Z E E E E E E E E E E E E
CDR 3
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
S S S S S S S S S S S S S S S S S S S S S S S S S
A A A A A A A A A A A A A A A A A A A A A A A A A
S S S S S S S S S S S S S S S S S S S S S S S S S
V V V V V V V V V V V V V V V V V V V V V V V V V
K K K K K K K K K K K K K K K K K K K K K K K K K
G G G G G G G G G G G G G G G G G G G G G G G G G
65 95 N Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y N Y D V D G D A D A D Y D Y N Y D A N Y GGY N Y D Y
i00 G S T G S S G N S G S S G S S G S S G S S G S S D Y P G S R G S S G S S K Y D Y G Y Y G S S Y G S Y G D S G S S G S S G S A D Y G N G S S ..... Y D G S Y D G S Y Y Y Y Y Y Y Y Y Y Y Y Y Y D Y D Y Y Y Y Y
a b c W Y F D W Y F D W Y F D W Y F D W Y F A W Y F D W Y F D W Y F D W Y F D W Y F D W Y F D W Y F D W Y V D W Y F D W Y F D W Y F D G Y F B W Y F D W Y F D W Y F D G Y F D W Y F D YYTMDY - Y W Y F D - H W Y F D Y Y Y -
Y Y Y Y Y Y Y H Y Y Y L D Y Y F Y Y Y G Y
lOt V V V V V V V V V V V V V V V V V V V V V V V V
same V,, germ line gene. It is notable that with only a few exceptions, the hapten contacting residues found in McPC603 are invariant, thus suggesting that the mode of interaction of these proteins with phosphocholine might be the same as m McPC603. TABLE II. - - Light chain hypervariable region sequences of routine phosphocholine-binding immunoglobulins.
MCPC603 SI07 MOPCI67 C57BL2857 MOPC511
24 K T R R R
S A S S S
S S S S S
CDR 27 a b c d e Q S L L N S G E S L Y S S K K S L L Y K K S L L Y K K S L L Y K -
1 f N H D D D
28 Q K K V G K G K G K
34 N H T T T
F Y Y Y Y
L L L L L
A A N N N
CDR 2 50 GASTRES GASNRYI LMSTRAS LMSTRAS LMSTRAS
56
CDR3 89 QNDHSYPLT AQFYSYPLT QQLVEYPLT QQLVEYPLT QQLVEYPLT
97
At first sight, the light chain sequences (table II) are quite different, forming three different classes [9.]. However, here again, the key residues, Tyr 94 and Leu 96, are invariant so that the general shape of the pocket probably remains intact. Asp 91 in McPC603 is replaced in the other light chain sequences by either Phe or Leu, thus losing the attractive effect of the negatively charged carboxylate ion. However, Asn 95 of the heavy chain in McPC603 is replaced in these other proteins by an aspartic acid, thus suggesting the possibility of a remarkable coupling of somatic mutations to maintain specificity. The restriction in the residues responsible for phosphocholine binding exhibited by these proteins may also exist in immunoglobulins of different specificities [8].
276
6e F O R U M D ' I M M U N O L O G I E
However, in the absence of a detailed three-dimensional analysis of a haptenimmunoglobulin complex, it is impossible to properly assess the conservation of the specificity determining features and it is only through a combination of sequence analysis with X-ray crystallographic studies that such conclusions can be reached.
Re[erences. [1] AMZEL, L. M., POLJAK, R. J., SAUL, F., VARGA, J. M. & RICHARDS, F. F., The three-dimensional structure of a combining region ligand complex of immunoglobulin N E W at 3.5 A resolution. Proc. nat. Acacl. Sei. (Wash.), 1974, 71, 1427-1430. [2] GEAHHART,P. J., JOHNSON, N. D., DOUGLAS,R. & HOOD, L., IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature (Lond.), 1981, 291, 29-34. [3] HENDRICKSON, W. A. & KONNERT, J. H., Incorporation of stereochemical information into crystallographic refinement, in ,( Computing in crystallography )>(R. Diamond, S. Ramaseshan & K. Venkatesan) (pp. 13.01-13.23). Indian Academy of Sciences, Bangalore, 1980. [41 KABAT, E. A., Wu, T. T., BILOFSKY, H., REID-MILLER, M. & PERRY, a . , (( Sequences of proteins of immunological interest ,,, U. S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1983. [5] MARIUZZA, R. A., JANKOVIC, D. L., BOULOT, G., AMIT, A. G., SALUDJIAN, P., LE GUERN, A., MAZIE, J. C. & POLJAK, R. J., Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal antilysozyme antibody and its antigen. J. tool. Biol., 1983, 170, 1055-1058. [6] PADLAN, E. A., SEGAL, D. M., SPANDE, T. F., DAVIES, D. R., RUDIKOFF, S. & POTTER, M., Structure at 4.5 A resolution of a phosphoryleholine-binding Fab. Nature (New Biol.) (Lond.), 1973, 145, 165-167. [7] RUDIKOVF,S., SATOW, Y., PADLAN,E., DAVIES, D. & POTTER, M., Kappa chain structure from a crystallized murine Fab: role of joining segment in hapten binding. Mol. Immunol., 1981, 18, 705-711. [8] RUDIKOVV, S., Immunoglobulin structure-function correlates: antigen binding and idiotypes, in ,( Contemporary topics in molecular immunology ,, (F. P. I n m a n & T. J. Kindt), 9 (p. 169), Plenum Publ. Co., New York, 1983. [9] SEGAL, D. M., PADLAN, E. A., COHEN, G. H., RUDIKOFF, S., POTTER, M. & DAVIES, D. R., The three-dimensional structure of a phosphorylcholine binding mouse immunoglobulin Fab and the nature of the antigen-binding site. Proc. nat. Acad. Sci. (Wash.), 1974, 71, 4298-4302. [1O] SILVERTON,E. W., DAVIES, D. R., SMITH-GILL, S. J. & POTTER, M., Crystallization of the Fab fragments of monoclonal antibodies to hen egg white lysozyme. Amer. Cryst. Ass. Abstracts, 1983, 11, No. 2, 28.
T H E Mcg L I G H T CHAIN: MULTIPLE CONFORMATIONS D E R I V E D FROM A SINGLE AMINO ACID SEQUENCE by A. B. E d m u n d s o n and K. R. Ely
Department o/Biology, University o/ Utah, Salt Lake City, UT 81112 ( U S A ) Introduction. In 1969, Harold F. Deutseh of the University of Wisconsin crystallized the serum IgG1 immunoglobulin and the urinary Benee-Jones (light chain) dimer from the patient Meg, who died of complications from amyloidosis. We subsequently produced various types of large crystals which were systematically subjected to X - r a y analysis.
271
[14] ROTHSTEIN, T. L. & GEFTER, M. W., Affinity analysis of idiotype-positive and [15] [16] [17]
[13] [19]
idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera. Mol. Immunol., 1983, 20, 161-168. I~UDIKOFF, S., GIUSTI, A. M., COOK, W. D. & SCHARFF, M. D., Single amino acid substitutions altering antigen-binding specificity. Proc. nat. Aead. Sci. ('Wash.), 1982, 79, 1979-1983. SHOENFELD, Y., ISENBERG, D. A., RAUCH, J., MADAIO, M. P., STOLLAR, B. D. & SCHWAnTZ, R. S., Idiotypie cross-reactions of monoclonal human lupus autoantibodies, d. exp. Med., 1983, 158, 718-730. TEILLAUD, J.-L., DESAYMARD, C., GUISTI, A. M., HASELTINE, B., POLLOCK, R. R., YELTON, D. E., ZACK, D. J. & SCHARFF, M. D., Monoclonal antibodies reveal the structural basis of antibody diversity. Science, 1983, 222, 721-726. THEOFILOPOULOS,A. N. & DIXON, F. J., Etiopathogenesis of murine SLE. lmmunol. Rev., 1981, 55, 179-216. TONEGAWX, S., Somatic generation of antibody diversity. Nalure (Lond.), 1983, 302, 575-581.
Acknowledgemenls. The work reported in this paper was supported by grants from the NIH (AI523L, AM32371. A1 10702), the NSF (PCM 836160), the ACS (IM-3LTC), a Cancer Center Grant (3PO CA1330) and the SLE Foundation. B. D. is an established investigator of the American Heart Association and N. C. is supported by a training grant (CA 09173) from the NCI.
ON THE S P E C I F I C I T Y OF A N T I B O D Y / A N T I G E N INTERACTIONS: P H O S P H O C H O L I N E B I N D I N G TO MePC603 AND THE CORRELATION OF T H R E E - D I M E N S I O N A L STRUCTURE AND SEQUENCE DATA
by E. A. Padlan, G. H. Cohen and D. R. Davies
Laboralory o/Molecular Biology, National Institute o[ Arthritis, Diabeies, and Digestive and Kidney Diseases, National Inslilules o/Health, Belhesda, MD, 20205 ( U S A ) Summary. Refined three-dimensional structures of McPC603 Fab and the complex with phosphocholine permit a detailed assessment of the residues crucial to determining the antibody specificity. Correlation with sequence data suggests that the structure of the binding site is highly conserved in immunoglobulins with phosphocholinebinding specificity. There is suggestive evidence that coupling of somatic mutations occurs to preserve antigen-binding specificity. The immune response is characterized b y specificity and diversity. While each antibody appears to be specific for a single antigen, the immune response can
272
6e FORUM D'IMMUNOLOGIE
generate up to 109 different specificities. In order to understand, at the molecular level, the nature of the interaction between antibody and antigen, it is necessary to have a high-resolution three-dimensional picture of the complex. Today it is possible to investigate antibody/antigen interactions directly by the crystallographic analysis of hybridoma products [5, 10]; in the past, structural studies were limited to myeloma proteins which, in some cases, could be shown to complex to certain haptens. Of the four Fab structures that have been determined by X-ray diffraction, only two have been demonstrated to bind hapten in the crystal. They are Fab NEW, which was shown to bind a vitamin K1 derivative [1] and McPC603, which binds to phosphocholine [6, 9]. During the last few years, the McPC603 Fab structure has been refined at 2.7 )k resolution and the complex of McPC603 Fab with phosphocholine has been refined independently at 3.1 )k. In this communication, we make a comparative analysis of the sequences of a number of mouse phosphocholine-binding immunoglobulins based on the refined structure of the phosphocholine-binding site in McPC603. Refinemenl ol the slruclures. The structure of Fab McPC603 has been refined by the application of Hendrickson-Konnert restrained least-squares procedure [3] combined with extensive modelling to a set of 2.7-h resolution intensity data collected photographically by oscillation methods (Satow, Cohen, Padlan and Davies, in preparation). The final R-value is 22.5~o with r. m. s. deviations from ideality of 0.02 A in bond lengths and 3.5 ~ in bond angles. The refined McPC603 Fab structure was used as the starting point for the refinement of the complex with phosphocholine (Padlan, Cohen and Davies, in preparation). The R-value after 12 cycles of HendricksonK o n n e r t refinement is 18.5% with r. m. s. deviations from ideality of 0.014 .~ in bond lengths and 2.8 ~ in bond angles; because of the lower resolution of this refinement, bond distance and angles were held more closely to the ideal values. Resulls. In figure 1 are shown the amino acid residues most closely in contact with phosphocholine in the combining site of McPC603. These residues are T y r 33, Arg 52, Asn 95 and Trp 100a from the heavy chain (the numbering scheme of K a b a t el al. [4] is used here). Light-chain contact residues include Asp 91, T y r 94 and Leu 96 together with main chain atoms of residue 92. The interactions between hapten and these residues involve extensive van der Waals contacts, hydrogen bonds and electrostatic interactions. The hydroxyl group of Tyr 33 is hydrogenbonded to one oxygen of the phosphate. The guanidinium group of Arg 52 is also within hydrogen-bonding distance of phosphate oxygens. The negative charge on the phosphate is to some extent neutralized by the positively charged guanidinium group of Arg 52. The positive charge of the choline moiety is partially neutralized by the side group of Asp 91 of the light chain. In addition to the residues that are in direct contact with the hapten, there are charged side groups that appear to play an ancillary role in the binding of phosphocholine. For example, buried deep in the hapten-binding cavity is Glu 35 of the heavy chain, which can serve to partially neutralize the positive charge of the choline as well as fix the position of T y r 94 of the light chain through a hydrogen bond [7]. Also, the charges on the heavy chain Glu 58 and the lysines at 52b, 54 and 64 could be influential in aligning the hapten as it approaches the cavity [Getzoff and Tainer, personal communication]. Sequence data are available for a number of murine phosphocholine-binding antibodies. In tables I and II are listed the hypervariable region sequences of the heavy and light chains of these proteins [4]. The heavy chain sequences of table I reflect the finding of Gearhart el al. [2] that these proteins are all derived from the
F 32
c~ F 3~
PC
FIG. 1. - -
Stereodrawing oI the combining site o[ .'llcPC603 with phosphocholine (PC) bound.
The lower residues (91-96 a n d F32) are from t h e l i g h t chain; t h e others are from t h e h e a v y chain
3-D STRUCTURE AND FUNCTION OF Ig
275
TABLE I. Heavy-chain hypervariable region sequences of routine phosphocholine-binding immunoglobulins. -
-
CDR 1
MCPC603 TEPCI5 HOPC8 SI07 SIO7.UI HPCMI P,PCM2 HPCM3 IIPCM6 "8PCG8 $63 Y5236 W3207 HPCGI4 MOPCSII HPCGI3 MOPCI67 CBBPC-3 C57BL 293 C57BL 1613 C57BL 2857 C57BL 23169 CBA/N IBSE5 CBA/J 6F9 CBA/J 7C6
CDR 2
31 35 D F Y M E D F Y M E D F Y M E D F Y M E D F Y M E D F Y M E DF YME DFYME DFYME D F Y M E DFYME D F Y M E D F Y M E A F Y M E B F Y M E D F Y M E D F Y M E D F Y M E D F Y H E D F Y M E D F Y M E D F Y Id E D F Y M E D F Y M E D F Y M E
50 52 A S R A S R A S R A S R A S R A S R ASRNK ASRNK ASRNK A S R ASRNK A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R A S R
a N N B N N N
b K K K K K K
N K N B N N N S N N N N N N N N
K K K K K K K K K K K K K K
c53 G N K A N D A N D A N D A N D A N D A N D A N D A N D A F D A N D A N D A N D A N D A N D V Y D A H D A N D A N D A N D A N D A N D A N D A N D A N D
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
T T T T T T T T T T T T T T T T R T T T T T T T T
T T T T T T T T T T T T T T T T T T T T T T T T T
E E Z E E E E E E E E E Z E E E E E E E E E E E E
CDR 3
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
S S S S S S S S S S S S S S S S S S S S S S S S S
A A A A A A A A A A A A A A A A A A A A A A A A A
S S S S S S S S S S S S S S S S S S S S S S S S S
V V V V V V V V V V V V V V V V V V V V V V V V V
K K K K K K K K K K K K K K K K K K K K K K K K K
G G G G G G G G G G G G G G G G G G G G G G G G G
65 95 N Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y N Y D V D G D A D A D Y D Y N Y D A N Y GGY N Y D Y
i00 G S T G S S G N S G S S G S S G S S G S S G S S D Y P G S R G S S G S S K Y D Y G Y Y G S S Y G S Y G D S G S S G S S G S A D Y G N G S S ..... Y D G S Y D G S Y Y Y Y Y Y Y Y Y Y Y Y Y Y D Y D Y Y Y Y Y
a b c W Y F D W Y F D W Y F D W Y F D W Y F A W Y F D W Y F D W Y F D W Y F D W Y F D W Y F D W Y F D W Y V D W Y F D W Y F D W Y F D G Y F B W Y F D W Y F D W Y F D G Y F D W Y F D YYTMDY - Y W Y F D - H W Y F D Y Y Y -
Y Y Y Y Y Y Y H Y Y Y L D Y Y F Y Y Y G Y
lOt V V V V V V V V V V V V V V V V V V V V V V V V
same V,, germ line gene. It is notable that with only a few exceptions, the hapten contacting residues found in McPC603 are invariant, thus suggesting that the mode of interaction of these proteins with phosphocholine might be the same as m McPC603. TABLE II. - - Light chain hypervariable region sequences of routine phosphocholine-binding immunoglobulins.
MCPC603 SI07 MOPCI67 C57BL2857 MOPC511
24 K T R R R
S A S S S
S S S S S
CDR 27 a b c d e Q S L L N S G E S L Y S S K K S L L Y K K S L L Y K K S L L Y K -
1 f N H D D D
28 Q K K V G K G K G K
34 N H T T T
F Y Y Y Y
L L L L L
A A N N N
CDR 2 50 GASTRES GASNRYI LMSTRAS LMSTRAS LMSTRAS
56
CDR3 89 QNDHSYPLT AQFYSYPLT QQLVEYPLT QQLVEYPLT QQLVEYPLT
97
At first sight, the light chain sequences (table II) are quite different, forming three different classes [9.]. However, here again, the key residues, Tyr 94 and Leu 96, are invariant so that the general shape of the pocket probably remains intact. Asp 91 in McPC603 is replaced in the other light chain sequences by either Phe or Leu, thus losing the attractive effect of the negatively charged carboxylate ion. However, Asn 95 of the heavy chain in McPC603 is replaced in these other proteins by an aspartic acid, thus suggesting the possibility of a remarkable coupling of somatic mutations to maintain specificity. The restriction in the residues responsible for phosphocholine binding exhibited by these proteins may also exist in immunoglobulins of different specificities [8].
276
6e F O R U M D ' I M M U N O L O G I E
However, in the absence of a detailed three-dimensional analysis of a haptenimmunoglobulin complex, it is impossible to properly assess the conservation of the specificity determining features and it is only through a combination of sequence analysis with X-ray crystallographic studies that such conclusions can be reached.
Re[erences. [1] AMZEL, L. M., POLJAK, R. J., SAUL, F., VARGA, J. M. & RICHARDS, F. F., The three-dimensional structure of a combining region ligand complex of immunoglobulin N E W at 3.5 A resolution. Proc. nat. Acacl. Sei. (Wash.), 1974, 71, 1427-1430. [2] GEAHHART,P. J., JOHNSON, N. D., DOUGLAS,R. & HOOD, L., IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature (Lond.), 1981, 291, 29-34. [3] HENDRICKSON, W. A. & KONNERT, J. H., Incorporation of stereochemical information into crystallographic refinement, in ,( Computing in crystallography )>(R. Diamond, S. Ramaseshan & K. Venkatesan) (pp. 13.01-13.23). Indian Academy of Sciences, Bangalore, 1980. [41 KABAT, E. A., Wu, T. T., BILOFSKY, H., REID-MILLER, M. & PERRY, a . , (( Sequences of proteins of immunological interest ,,, U. S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1983. [5] MARIUZZA, R. A., JANKOVIC, D. L., BOULOT, G., AMIT, A. G., SALUDJIAN, P., LE GUERN, A., MAZIE, J. C. & POLJAK, R. J., Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal antilysozyme antibody and its antigen. J. tool. Biol., 1983, 170, 1055-1058. [6] PADLAN, E. A., SEGAL, D. M., SPANDE, T. F., DAVIES, D. R., RUDIKOFF, S. & POTTER, M., Structure at 4.5 A resolution of a phosphoryleholine-binding Fab. Nature (New Biol.) (Lond.), 1973, 145, 165-167. [7] RUDIKOVF,S., SATOW, Y., PADLAN,E., DAVIES, D. & POTTER, M., Kappa chain structure from a crystallized murine Fab: role of joining segment in hapten binding. Mol. Immunol., 1981, 18, 705-711. [8] RUDIKOVV, S., Immunoglobulin structure-function correlates: antigen binding and idiotypes, in ,( Contemporary topics in molecular immunology ,, (F. P. I n m a n & T. J. Kindt), 9 (p. 169), Plenum Publ. Co., New York, 1983. [9] SEGAL, D. M., PADLAN, E. A., COHEN, G. H., RUDIKOFF, S., POTTER, M. & DAVIES, D. R., The three-dimensional structure of a phosphorylcholine binding mouse immunoglobulin Fab and the nature of the antigen-binding site. Proc. nat. Acad. Sci. (Wash.), 1974, 71, 4298-4302. [1O] SILVERTON,E. W., DAVIES, D. R., SMITH-GILL, S. J. & POTTER, M., Crystallization of the Fab fragments of monoclonal antibodies to hen egg white lysozyme. Amer. Cryst. Ass. Abstracts, 1983, 11, No. 2, 28.
T H E Mcg L I G H T CHAIN: MULTIPLE CONFORMATIONS D E R I V E D FROM A SINGLE AMINO ACID SEQUENCE by A. B. E d m u n d s o n and K. R. Ely
Department o/Biology, University o/ Utah, Salt Lake City, UT 81112 ( U S A ) Introduction. In 1969, Harold F. Deutseh of the University of Wisconsin crystallized the serum IgG1 immunoglobulin and the urinary Benee-Jones (light chain) dimer from the patient Meg, who died of complications from amyloidosis. We subsequently produced various types of large crystals which were systematically subjected to X - r a y analysis.