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ANTIBODIES AND ANTIGENS
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protein molecule,58-6° these methods should increasingly provide both new insight into the eonformational and dynamic features of antigen specificity, and give a viable alternative to X-ray crystallographic methods, which ultimately rely on the availability of suitable crystals for analysis. In realizing their full potential, one would predict that over the next few years a rapid growth will be seen in the structural database relating to antibodypeptide interactions, and a concurrent improvement in our general level of understanding of the molecular basis for one of the natural recognition processes. 5s L. Riechmann, J. Foote, and G. Winter, J. Mol. Biol. 203, 825 (1988). 59 A. Skerra and A. Pluckthun, Science 240, 1038 (1988). 60 H. Field, G. T. Yarrington, and A. R. Rees, Protein Eng. 3, 641 (1989).
[10] N u c l e a r M a g n e t i c R e s o n a n c e f o r S t u d y i n g Peptide-Antibody Complexes by Transferred Nuclear Overhauser Effect Difference Spectroscopy By JACOBANGLISTERand FRED NAIDER Introduction Short synthetic peptides have been useful as probes for the antigenic structure of proteins.~ Immunization with a flexible synthetic peptide that is homologous in amino acid sequence with a segment of a protein elicits the production of a spectrum of antibodies. These polyclonal antibodies may recognize different conformations of the peptide and only a fraction of these antibodies may bind the native protein. We have initiated a detailed analysis of the interaction of an immunizing peptide with monoclonal antibodies that either do or do not recognize the native protein. Studies of such complexes should provide insights into antibody function and selectivity. The principal aims of our research are (1) to study the molecular basis of specific antibody-peptide interaction and to understand how the diversity in the amino acid sequence of the antibody affects combining site structure, antibody affinity, and specificity; and (2) to characterize the different antigenic structures of peptide antigens, and to relate peptide conformation to the production of antibodies cross-reactive with the parent protein. H. J. Dyson, R. A. Lerner, and P. E. Wright, Annu. Rev. Biophys. Chem. 17, 305 (1988).
METHODS IN ENZYMOLOGY,VOL. 203
Copyright© 1991by AcademicPrizes,Inc. All rightsof reproductionin any form reserved.
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In order to learn about the molecular bails for the cross-reactivity of antibodies with proteins and peptides derived from them it is necessary to determine the three-dimensional structure of peptide-antibody complexes. Although crystal structures are available for several Fab fragments, only limited success has been achieved on Fab-peptide or Fv-peptide complexes) In principle, nuclear magnetic resonance (NMR) spectroscopy can be used to study the three-dimensional structure of antibody-peptide complexes in solution. Complete structure determination by this technique, however, is currently limited to small proteins of up to 150 amino acid residues. 3-5 Studies on larger proteins are hindered by the loss of spectral resolution, which stems from resonance broadening due to enhanced relaxation pathways and overlap of individual resonances from the increased number of protons. Consequently very few N M R studies of large proteins have been reported. This chapter describes an approach to studying ligand-protein interaction using two-dimensional (2D) transferred nuclear Overhauser effect (NOE) difference spectroscopy. The interaction of antibodies with peptide antigens provides a specific case study. Methodology The antibody molecule has a molecular weight of about 150,000 and contains more than 6000 nonexchangeable protons. To obtain the simplest proton N M R spectrum possible, it is necessary to work with the smallest fragment of the antibody that retains its affinity for the antigen. This fragment is the antibody Fv, which contains about 220 amino acids and has a molecular weight of approximately 25,000. 6 The Fv fragment was used by Dwek and co-workers in their pioneering N M R studies of the antibody MOPC315 complexed with a dinitrophenyl hapten. 7,s Unfortunately the Fv can be obtained by proteolytic cleavage only for a very limited number of antibodies. Therefore, until recently, only the larger Fab fragment was readily available for N M R and crystallographic studies. In addition to the variable domains, the Fab contains one constant domain from each chain. The molecular weight of the Fab is about 50,000. It 2R. L. Stanfield, T. M. Fieser, R. A. Lerner, and I. A. Wilson, Science 242, 712 (1990). 3K. Wfithrich, "NMR of Proteins and NucleicAcids." Wiley,New York, 1986. 4K. WOthrich,Ace. Chem. Res. 22, 36 (1989). 5D. A. Torchia, S. W. Sparks, and A. Bax, Biochemistry 2.8,5509 (1989). 6j. Hochman, D. Inbar, and D. Givol, Biochemistry 12, 1130(1973). 7S. K. Dowerand R. A. Dwek, in "BiologicalApplications of Magnetic Resonance" (R. G. Shulman, ed.), p. 271. AcademicPress, New York, 1979. 8R. A. Dwek, J. C. A. Knott, D. Marsh, A. C. MeLaughlin, E. M. Press, N. C. Price, and A. I. White, Eur. J. Biochem. 53, 25 (1975).
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ANTIBODIES AND ANTIGENS
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contains about 440 amino acids and more than 2000 nonexchangeable protons. Genetic engineering techniques have been used to express the Fv in myeloma cells and Escherichia coli, 9-1~ and it is expected that future NMR studies will take advantage of the increased resolution expected from studies on Fv-antigen complexes (see Wright et al. 12 for a preliminary NMR study on an Fv fragment).
Deuterium Labeling of Antibody The ~H NMR spectrum of the Fab is very poorly resolved even when measured on a 500- or 600-MHz spectrometer. In order to simplify the spectrum and assign resonances of the Fab to specific amino acid types the antibody is labeled with deuterated amino acids.13 This biosynthetic labeling is accomplished by feeding hybridoma cells producing the monoclonal antibody a controlled diet containing selected deuterated amino acids. In studies of antibody-antigen interactions it is beneficial to concentrate on simplifying the spectrum of the aromatic protons for three reasons: (1) that region of the spectrum is a priori less crowded; (2) all aromatic hydrogens can be very efficiently labeled; and (3) aromatic amino acids have a major role in antibody-antigen interactions, with solvent exclusion contributing significantly to the binding energy.14
Two-Dimensional Transferred Nuclear Overhauser Effect Difference Spectroscopy Three-dimensional structure determination by NMR is based on the observation of magnetization transfer between neighboring protons due to the NOE. 15 The magnitude of the NOE is dependent on the reciprocal of the sixth power of the distance between interacting nuclei and is observed up to a distance of approximately 5 A. A two-dimensional (2D) NOE experiment (NOESY) contains the information about magnetization transfer due to the NOE between all pairs of neighboring protons in the molecule.3 Despite the power of 2D NOE spectroscopy this technique has not been applied to the study of the structure of large proteins because of 9 A. Skerra and A. Plueckthun, Science 240, 108 (1988). ~oL. Riechmann, J. Foote, and G. Winter, J. Mol. Biol. 203, 825 (1988). II R. E. Bird, K. D. Hardman, J. W. Jaeobson, S. Johnson, B. M. Kaufman, S.-M. Lee, T. Lee, S. H. Pope, G. S. Riordan, and M. Whitlow, Science242, 423 (1988). 12p. E. Wright, H. J. Dyson, R. A. Lerner, L. Riechmann, and P. Tsang, Biochem. Pharmacol. 40, 83 (1990). 13j. Anglister, T. Frey, and H. M. McConnell, Biochemistry 23, 1138 (1984). ,4 E. A. Padlan, Proteins: Funct. Struct. Genet. 7, 112 (1990). ~5j. H. Noggle and R. E. Sehirmer, "The Nuclear Overhauser Effect: Chemical Applications." Academic Press, New York, 1971.
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the poor spectral resolution found in 2D NMR spectra of these macromolecules. Specific deuteration of aromatic amino acids significantly improves the resolution in the NOESY spectrum; however, cross-peak overlap still prevents resonance identification.~ In order to discern protein-fig,and interactions and the conformation of the bound ligand using the NOESY spectrum of the complex, one must discriminate between cross-peaks due to intermolecular interactions and the numerous cross-peaks due to intramolecular interactions in the complexed protein. Most pertinent is the fact that it is almost impossible to identify the bound ligand resonances, since they are at least as broad as the protein resonances and often considerably broader due to a fast ligand off-rate. Therefore the bound ligand resonances are unresolved from the background. To circumvent this problem we take advantage of a phenomenon known as transferred nuclear Overhauser effect spectroscopy (TRNOE). ~,18 This phenomenon is observed when the ligand molecules exchange rapidly between the bound and the free state. Under conditions where the ligand off-rate is fast relative to the TI relaxation time of both the Fab and the ligand protons, and to the mixing time used in the NOESY experiment, the spectrum of the Fab in the presence of excess ligand (excess spectrum) contains TRNOE cross-peaks. These cross-peaks reflect magnetization transfer that occurs between protons in the bound state. Transfer can be between Fab protons and protons of the bound ligand and/or between different protons of the bound ligand. This transferred magnetization is observed via the free ligand due to the relatively rapid exchange between the bound and free fig,and. These inter- and intramolecular TRNOE cross-peaks are accompanied by numerous cross-peaks due to intramolecular interactions between Fab protons. Since there is essentially no free ligand, none of the TRNOE cross-peaks appears in the NOESY spectrum of the Fab saturated with ligand at a 1 : 1 molar ratio (saturated spectrum). However, the numerous cross-peaks due to intramolecular interactions in the saturated Fab are the same as those in the NOESY spectrum of the Fab in the presence of a ligand excess. By subtracting the saturated spectrum from the excess spectrum, one obtains a difference spectrum in which only TRNOE cross-peaks are observed, t9 This TRNOE difference spectrum is usually well resolved. Its resolution may be further improved by specific deuteration, which simultaneously allows the assignment of cross-peaks to a specific type of amino acid. 16j. Anglister, Q. Rev. Biophys. 23, 173 (1990). ~7P. Balaram, A. A. Bothner-By, and E. Breslow, J. Am. Chem. Soc. 94, 4017 (1972). t8 j. p. Albrand, B. Birdsall, J. Feeney, G. C. K. Roberts, and A. S. V. Burgen, Int. J. Biol. Macromol. 1, 37 (1979). 19j. Anglister, R. Levy, and T. Seherf, Biochemistry 28, 3360 (1989).
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It should be noted that intramolecular NOE in small molecules has a positive sign, with intensity decreasing as a function of molecular weight. For molecules having a molecular weight of about 1500 the NOE is vanishingly small or zero. For larger molecules it becomes negative, and the intensity increases to a maximum o f - 1 with increasing molecular weight. Therefore, in the presence of a binding protein, the sign of transferred NOE cross-peaks due to intramolecular interactions in the bound ligand usually will be negative. The exact dependence of the TRNOE intensity as a function of the binding constant, ligand off-rate, molar ratio between ligand and protein, averaging of the resonances of bound and free ligand, and the cross-relaxation rate in the free ligand is thoroughly described by Clore and Gronenborn. 2°,2~
Assignment of TRNOE Cross-Peaks Cross-peaks appearing in the TRNOE difference spectrum arise from the following: (1) exchange between bound and free ligand, (2) magnetization transfer between antibody protons and free figand protons via the bound state, (3) intramolecular magnetization transfer within the bound ligand via exchange with the free. Therefore at least one of the two frequency values of a TRNOE cross-peak should be of a free ligand proton, while the corresponding second resonance is (1) the same proton of the bound ligand, (2) an antibody proton or (3) another proton of the free ligand. The assignment of cross-peaks to their respective free ligand protons is based on the 2D homonuclear correlated spectrum (COSY) of the ligand, t9 If changes in chemical shift of the ligand protons on binding are large relative to the fig,and off-rate, averaging of bound and free ligand proton resonances is prevented and the COSY spectrum of the ligand can be used. However, for fast off-rates, resulting in averaging of the resonances of the bound and free ligand, one should measure the COSY spectrum of the Fab solution in the presence of the same ligand excess used for the NOESY measurements. Contributions of the Fab to the COSY spectrum mostly vanish due to the linewidth of the Fab protons, and only the contributions of the much smaller ligand are observed. When the resonance of a ligand proton interacting with the antibody falls in a region where a few ligand resonances overlap, unambiguous assignment requires repetition of the experiment with a specifically deuterated ligand. 20 G. M. Clor¢ and A. M. Gronenborn, J. Magn. Reson. 48, 402 (1982). 2~ G. M. Clore and A. M. Gronenborn, J. Magn. Reson. 53, 423 (1983).
[ I 0]
NMR FOR STUDYING PEPTIDE-ANTIBODY COMPLEXES
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FIG. 1. Two-dimensional difference spectra between the NOESY spectrum of TE32 Fab with a fourfold excess of the peptide CTP3 (VEVPGSQHIDSQKKA) and the NOESY spectrum of the ~ptide-saturated Fab, showing interactions of specific types of antibody aromatic residues with peptide residues. Assignment to antibody residues is marked by capital letters and arbitrary numbers, while assignment to ~ptide residues is marked by lower-case letters and their location in the sequence. (A) Interactions of antibody tyrosine and laistidine residues with peptide residues. Antibody phenylalanine and tryptophen residues are perdeuterated, while tyrosine residues are deuterated at 2,6-phenyl positions. (B) Interactions of antibody tryptophan and histidinc residues with peptidc residues. Antibody phenylalanine and tyrosine residues are perdeuterated. (C) Interactions of antibody phenylalanine and histidine residues with peptide residues. Antibody tyrosine and tryptophan residues are perdeuterated.
Our studies concentrate on the interactions between aromatic residues of the antibody and the amino acids of a peptide of cholera toxin (CTP3). CTP3 (VEVPGSQHIDSQKKA) 2ta contains only one aromatic residue, histidine, and the resonances of the free peptide histidine can be easily identified. When interaction between a nonaromatic peptide proton and an aromatic proton is observed, and the chemical shifts of the aromatic protons differ from those of the peptide histidine imidazole protons, it is assigned to antibody aromatic residue. The assignment to a specific type of amino acid is based on specific deuteration of the aromatic amino acids of the antibody, t9 Figure 1 shows the assignments of the TRNOE cross-peaks 2~aSingle-letter abbreviations used for amino acids: A, alanine; R, arglnine; N, asparaglne; D, aspartic acid; C, cysteine; Q, glutamine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine.
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234
ANTIBODIES AND ANTIGENS
[ 10]
to the aromatic residues of the TE32 anti-CTP3 antibody based on the measurements of the 2D TRNOE difference spectrum for three preparations of Fab differing in the labeling of antibody aromatic residues. When one of the two chemical shifts of a cross-peak is of a free ligand proton and the difference between the two is less than about 2 ppm, this cross-peak may be due to exchange. Assignment of such cross-peaks is facilitated by repeating the measurements at different temperatures. At lower temperature the Fab resonances become broader, whereas those of the bound peptide become narrower due to a slower off-rate. Such narrowing can be easily detected if the off-rate is on the order of or faster than the reciprocal of the intrinsic /'2 of the protons in the complex, when both chemical shifts of a cross-peak in the TRNOE difference spectrum are the same as those of the free ligand protons and the line structure of the two resonances involved is observed, this cross-peak is assigned to intramolecular interaction in the bound ligand.
Experiments Involving Slow Ligand Off-Rate When the ligand off-rate is slow relative to the mixing period used in the NOESY experiment and relative to the spin-lattice relaxation time of the Fab and peptide protons, the TRNOE becomes much weaker or nonobservable. We encountered this problem while studying the interactions between the CTP3 peptide and the TE34 antibody.22 In order to be able to use TRNOE difference spectroscopy we searched for minor modifications of the peptide that would decrease the binding constants and enhance its off-rate. We found that conversion of the C-terminal carboxyl into an amide decreases the binding constant of the peptide to TE34 by two orders of magnitude while increasing its off-rate similarly. Indeed, TRNOE difference spectra with excellent signal-to-noise ratio were obtained using the modified peptide. To verify that this modification did not alter the interactions of the antibody with the unmodified portion of the peptide we repeated the experiments with a truncated peptide, acetyI-IDSQKKA, corresponding to residues 9 to 15 of CTP3. This peptide analog lacks one residue (His-8) from the epitope found to be recognized by TE34; however, it contains an intact C-terminal carboxyl. The binding constant of this peptide analog to TE34 is only one order of magnitude less than that for CTP3 binding to the antibody. Our experiments show that both CTP3 with an amide at the carboxyl terminus and the truncated analog exhibit very similar cross-peaks. Thus, we conclude that minor modification of the epitope recognized by the antibody does not significantly affect antibody interaction with the remainder of the epitope. 22 The use of modified peptides that overlap the antigenic epitope permits study of the interac-
[10]
N M R FOR STUDYING PEPTIDE-ANTIBODY COMPLEXES
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FIG. 2. Two-dimensional TRNOE difference spectra obtained after specific chain labeling. (A) Interactions of the light-chain aromatic residues of TE34 Fab with the NCA peptide (Ae-VEVPGSQHIDSQKKA-NH2). (B) Interactions of heavy-chain aromatic residues with the NCA peptide. (C) Interactions of aromatic residues from both chains with the NCA peptide. Assigned antibody residues are marked by capital letters and arbitrary numbers; peptide protons are marked by small letters and numbers denoting the location in sequence of the residues to which the protons belong.
tions between the antibody and the whole peptide. The strategy that we developed can be used to extend the applicability of TRNOE difference spectroscopy to the study of other protein-ligand complexes involving slow ligand off-rates.
Chain Labeling Antibody light and heavy chains can be separated and later recombined to reconstitute the native conformation of the antibody. 23,24This procedure can be used to label each of the chains and subsequently assign resonances to a specific chain. 25 For example, two differently labeled Fab samples (one deuterated, the other not deuterated) are prepared, the interchain disulfide bond is reduced and alkylated, and the chains separated by ion-exchange chromatography under denaturing conditions. The deuterium-labeled heavy-chain fragment is recombined with the unlabeled fight chain and vice versa. The native conformation is then recovered by dialyzing away the denaturant. Figure 2 shows the TRNOE difference spectra obtained for reconstituted Fab in which the chains were specifically labeled.26Figure 2A 22j. Anglister and B. Zilber, Biochemistry 29, 921 (1990). F. Franek and R. S. Nezlin, Folia Microbiol. 8, 128 (1963). H. Metzger and M. Mannik, J. Exp. Med. 120, 765 (1964). 25j. Anglister, T. Frey, and H. M. McConnell, Nature (London) 315, 65 (1985). 26 B. Zilber, T. Scheff, M. Levitt, and J. Anglister, Biochemistry 29, 10032 (1990).
236
ANTIBODIES AND ANTIGENS
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shows the interactions of the aromatic protons of the light chain and Fig. 2B shows the interactions of the aromatic protons of the heavy chain. Figure 2C was obtained for native unlabeled Fab and shows the interactions of aromatic protons from both chains. Figure 2C correspond very well to the sum of the spectra presented in Fig. 2A and B. Assignment of the Antibody Interactions to Specific Residues The assignment of TRNOE cross-peaks allows us to define interactions between various types of amino acids. Given the present state of the art it is impossible to make a complete structure determination of a Fab fragment using NMR spectroscopy. Thus, we are confronted with the problem of extending a given NMR assignment from a type of amino acid to a given residue in the Fab. This problem has been approached using computer model building and conformational energy calculations to generate a starting model for the NMR studies. It was first used by Dwek and his coworkers to study the combining site of an anti-DNP antibody. 7,8 The computerized model building is based on principles introduced by Padlan et aL27 This procedure takes advantage of the homology in the three dimensional structure between various antibodies, which is especially high in the conserved regions that form the immunoglobulin fold. The main obstacle is the modeling of the hypervariable loops, and different approaches to this problem have been developed.28 Using the calculated models, we can judge what amino acids in the combining site are exposed to the solvent and, therefore, have the potential to interact with the antigen. By combining the NMR information, the amino acid sequence data of the six CDRs and the preliminary model, most ligand-protein interactions can be assigned to specific antibody residues. Additional information about interresidue interactions in the antibody combining site can facilitate the assignment of the interactions to the specific residues. Such information can be obtained by calculating the difference between the excess spectrum and the NOESY spectrum of the Fab itself. This difference spectrum contains cross-peaks due to interactions with the antigen and those due to intra-Fab interactions of antibody protons that change their chemical shift on antigen binding.
27 E. A. Padlan, D. R. Davies, I. Pecht, D. Givol, and C. Wright, Cold Spring Harbor Syrup. Quant. Biol. 41, 627 (1976). 2s C. Chothia, A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip, P. M. Coleman, S. Spinelli, P. M. Alzari, and R. J. Poljak, Nature (London) 342, 877 0989).
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Derivation of Constraints on Proton-Proton Distances The 2D TRNOE difference spectrum contains cross-peaks due to all the short-range interactions (proton-proton distance < 4 ~,) between the antibody and the peptide. Measurements of the intensity ofintramolecular magnetization transfer between a pair of neighboring protons after a short irradiation period of one of them or after a short mixing time in a NOESY experiment are used to evaluate the distance between the protons. Such measurements have been carded out to obtain constraints on protonproton distances that were subsequently used to calculate the threedimensional structure of small proteins. 3 Clore and Gronenborn extended the application of such measurements to transferred NOE in proteinligand complexes. 20,2~ Their analysis distinguishes three regions of chemical exchange. In the case of fast exchange between bound and free ligand, when the ligand off-rate is faster then 10 times the spin-lattice relaxation time of its protons and when the resonances of the bound and free ligand are averaged, the magnitude of the transferred NOE (Nn~) between an antibody proton, A, and a peptide ligand proton, H, is given by Nn.A (1 -- a)an~ rm, where trm,~is the cross-relaxation rate between an antibody proton and neighboring bound peptide proton, a is the mole fraction of the free peptide, and rm is the mixing time in the NOESY experiment or the irradiation time in a 1D NOE experiment. This simple relationship applies to both 1D and 2D TRNOE experiments. The distance ratios between different pairs of protons in the complex can be determined from trrtA, which is inversely proportional to the sixth power of the distance between the protons. When the chemical shifts of the bound and free peptide resonances are not averaged but the off-rate is still faster than T1 and ana, and the peptide is present in large molar excess, the TRNOE intensity between an antibody proton and a free peptide proton is still approximated by the above equation. In the case of medium off-rate the efficiency of the TRNOE will depend on the off-rate 2°,21 and the above equation for Nn,~ can serve only as an upper limit. If the exchange between bound and free ligand is slow on the cross-relaxation and spin-lattice relaxation scale the transferred NOE intensity is vanishingly small. To translate cross-peak intensities into restraints on proton-proton distances one needs a calibration usually obtained by measuring the intensity of a cross-peak between a pair of protons separated by a known and fixed distance. To obtain such calibration we measured the difference spectrum between the NOESY spectrum of TE34 Fab in the presence of 10-fold peptide excess and the NOESY spectrum of the uncomplexed Fab (without spectrum). In addition to cross-peaks due to transferred NOE this
238
ANTIBODIES AND ANTIGENS
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type of difference spectrum shows cross-peaks due to intra-Fab interactions of protons experiencing changes in chemical shift on antigen binding. When all tryptophen and phenylalanine residues ol~ the antibody were deuterated while tyrosine residues were unlabeled the difference spectrum measured for the Fab contains very well-resolved cross-peaks due to intmresidue interactions between the ~ and Jl and between the ~2 and J2 tyrosine protons. These cross-peaks are not observed when the C6 protons of the antibody tyrosine residues are deuterated. The intensity of the resolved cross-peaks is then compared to the intensity of the exchange cross-peak of Ca H of the peptide histidine. Since the exchange cross-peak appears in all the TRNOE difference spectra, and all spectra are measured at the same mixing time and with the same molar ratio of peptide, this cross-peak can serve as an internal standard for calibrating the intensities in all TRNOE difference spectra. Through this internal standard one gets the ratio between cross-peak intensities in the TRNOE difference spectrum and the cross-peak due to intratyrosine interactions and subsequently the ratio between the distances of the corresponding pairs of protons. This analysis indicates that all the cross-peaks due to intermolecular interactions observed in the TRNOE difference spectra are due to strong interactions between protons that are less than 3.5 A apart.
Experimental
Procedures
Sample Preparation Hybridoma cells producing antibodies are grown as monolayers in 700 ml T flasks on RPMI 1640 medium29 supplemented with only 2% fetal calf serum to increase the efficiency of the labeling. Supernatant is collected every 2 - 3 days and 50 ml of fresh medium is supplied to each flask. Antibody production is dependent on the specific cell line and varies between 20 and 60 mg/liter. Biosynthetic specific deuteration of the antibody is accomplished by feeding the hybridoma cells producing the antibodies medium containing the selected deuterated amino acids according to the RPMI recipe. Antibodies are purified utilizing protein A-Sepharose CL-4B column chromatography)°,3t Antibody solution is concentrated by an Mr 25K cutoffcollodion bag (Schleicher & Schuell, Dassel, Germany) to ZgG. E. Moore, R. E. Gerne, and H. A. Franklin, J. Am. Med. Assoc. 199, 519 (1967). GIBCO Laboratories, Grand Island, New York, Technical Manual, 1982. ~oG. Otting, H. Widmer, G. Wagner, and K. WOthrich, J. Magn. Resort. 66, 187 (1986). 3~V. T. Oi and L. A. Herzenberg, in "Selected Methods in Cellular Immunology" (B. B. Mishell and S. M. Shiigi, eds.), p. 351. Freeman, San Francisco California, 1980.
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239
a concentration of 30 mg/ml. Fab is obtained by papain digestion32 and purified by applying the reaction mixture to a Sephadex G-100 column linked in series to a protein A column (50 ml protein A-Sepharose CL-4B/ 200 mg of antibody). The columns are preequilibrated and developed with 50 m M Tris-HC1, pH 9, containing 0.15 M sodium chloride and 0.05% (w/v) sodium azide. The large peak of Fab is preceded by a much smaller peak of (Fab')2. The Fc binds to the protein A and is eluted with citrate buffer, pH 4.5. The Fab is concentrated to 3 m M with a collodion bag and the aqueous solution is dialyzed against four changes of 10 m M phosphate-buffered dueterium oxide, pH 7.1, containing 0.05% sodium azide. A typical NMR sample contains 500/tl of 3 m M Fab. Antibodies are never lyophilized or frozen. NMR Measurements
NOESY spectra are measured on a 500-MHz spectrometer in the phase-sensitive mode using a proton-selective probe. A mixing time of 100 msec, which was found to be optimal for obtaining maximum intensities for the strong cross-peaks observed, is used. The carrier frequency is set on the HDO line, and a spectral width of 6000 Hz is used. The HDO line is presaturated, using minimal power, for 3 see before the first 90* pulse is applied. Spectra are Fourier transformed in both dimensions after application of a squared cosine window function. A considerable reduction in tl ridges is achieved by using the method of Otting et aL) ° already incorporated in the standard 2D Fourier transformation in Bruker's software. Spectra are not symmetrized. The two spectra used in the difference calculation are measured consecutively, Fab concentration is between 2 and 3.5 m M in 0.01 M phosphate-buffered 1:)2O, pD 7.15, and the temperature is 42 °. Spectra are recorded with 256 values of q ; for each value, 64 or 80 scans are collected that are preceded by 4 dummy scans. Measurements of the TRNOE difference spectra for 60- and 100-msec mixing times exclude the possibility that the cross-peaks observed in the TRNOE difference spectra at 100 msec are due to spin diffusion. Difference Spectra Calculations
The phases of the initial measurements (first tl value) of both 2D experiments are matched to obtain a 1D difference with minimal distortion to resonances and baseline. This is accomplished first by visual comparison of the two spectra and then by fine numerical phase correction of 32 R. R. Porter, Biochem. J. 73, 119 (1959).
240
ANTIBODIES A N D ANTIGENS
[10]
one to match the other. The phase corrections thus obtained are applied to the preliminary 2D Fourier transformation. From the NOESY spectrum of the Fab with excess peptide, we select a row crossing a free peptide resonance. The same row is selected from the NOESY spectrum of the peptide-saturated Fab, and the two are subtracted. The phase of the resulting 1D difference spectrum is numerically corrected to obtain a pure LorentzJan for the resonance of the peptide proton. This phase correction is now used to reprocess the two NOESY spectra, and the difference between these rows is reexamined to verify that the resonance of the free peptide retains a pure Lorentzian shape. This procedure ensures the matching of phases between the two 2D spectra in the region containing cross-peaks between nonaromatic protons. The rows and columns crossing the diagonal at the resonances of the histidine imidazole protons of the peptide require additional slight phase correction. Baseline distortions due to t2 ridges are corrected for by subtracting from the 2D difference spectrum the value for a column with no signal at the intersection with the diagonal of the 2D spectrum. Further baseline correction of individual rows is carried out where necessary by fitting a baseline with a fourth-order polynomial.
Conclusions and Perspectives The methodology developed in our laboratory allows one to obtain detailed information concerning the interactions between residues in a peptide antigen and residues in the combining site of a monoclonal antibody. Transferred NOE difference spectroscopy, in conjunction with specific deuteration of the antibody, provides parameters that give information about distances between the above residues. Unfortunately, the number of NOE cross-peaks are not sufficient to calculate an unequivocal structure for the bound antigen. To circumvent this problem we apply models for the antibody combining site that are predicted based on structures for CDRs determined by X-ray crystallography. These models help us to verify assignment of the peptide-antibody interactions and can be used along with NOE distance constraints, energy minimization, and molecular dynamics calculations to provide a 3D model of the structure of the bound peptide antigen. Moreover, comparison of NMR data with calculated models allows one to examine the validity of a given model and to exclude "bad models" that result from choosing improper segments from the known three-dimensional structures of other antibodies. It is important to emphasize that the above approaches are warranted by the severe overlap problem that is confronted in studies of the interaction of peptides with large proteins. This problem has been encountered by others and elegant
[ 11 ]
ISOTOPE-EDITEDNMR
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procedures involving isotope editing 33,34 and 2D difference spectroscopy with deuterated ligands have been developed. 35 In all cases to date, information forthcoming from such studies requires comparison with crystal structures to rationalize interactions that are apparent in the N M R spectra. The combined application o f 2D T R N O E difference spectroscopy and isotope editing procedures holds great promise for unraveling the mysteries o f p e p t i d e - p r o t e i n interactions. At present, despite some obvious shortcomings, these are the only approaches to study the conformation o f peptides b o u n d to antibodies, enzymes, or receptors in solution. Acknowledgments We are most grateful to Michael Levitt, Rina Levy, Taft Scherf, and Barbara Zilber for their important contributions to the work describedin this chapter. This work was supported by the United States-Israel Binationfl ScienceFoundation. F. N. is a visiting professoron a Fulbright International ExchangeProgram. 33S. W. Fesik, J. R. Luly, J. W. Erickson, and C. Abad-Zapatero, Biochemistry 27, 8297 (1988). P. Tsang, T. M. Feiser, J. M. Ostresh, R. A. Houghten, R. A. Lerner, and P. E. Wright, Fronteirs NMR Mol. Boil., 63 (1990). 32S. W. Fesik and E. R. P. Zuiderweg,J. Am. Chem. Soc. 111,5013, (1989).
[11] Isotope-Edited Nuclear Magnetic Resonance Studies of F a b - P e p t i d e Complexes
By P. TSAN6,M. RANCE,and P. E. WRmHT Introduction Through the development o f isotope-edited techniques, 1-3 the application o f nuclear magnetic resonance ( N M R ) spectroscopy to studies o f higher molecular weight systems such as F a b - a n t i g e n or F a b ' - a n t i g e n complexes has become feasible. 4-7 The general concept and principles o f this m e t h o d will be described briefly in this chapter, and illustrated with results obtained in our laboratory. R. H. Griffeyand A. G. Redfield, Q. Rev. Biophys. 19, 51 (1987). 2A. Bax, S. W. Sparks, and D. A. Torchia, this series, Vol. 176, p. 134. 3G. Wagner, this series, Vol. 176, p. 93. 4 p. Tsang, T. M. Fieser, J. M. Ostresh, R. A. Lerner, and P. E. Wright, Pept. Res. 1, 87 (1988).
METHODS IN ENZYMOLOGY, VOL. 203
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protein molecule,58-6° these methods should increasingly provide both new insight into the eonformational and dynamic features of antigen specificity, and give a viable alternative to X-ray crystallographic methods, which ultimately rely on the availability of suitable crystals for analysis. In realizing their full potential, one would predict that over the next few years a rapid growth will be seen in the structural database relating to antibodypeptide interactions, and a concurrent improvement in our general level of understanding of the molecular basis for one of the natural recognition processes. 5s L. Riechmann, J. Foote, and G. Winter, J. Mol. Biol. 203, 825 (1988). 59 A. Skerra and A. Pluckthun, Science 240, 1038 (1988). 60 H. Field, G. T. Yarrington, and A. R. Rees, Protein Eng. 3, 641 (1989).
[10] N u c l e a r M a g n e t i c R e s o n a n c e f o r S t u d y i n g Peptide-Antibody Complexes by Transferred Nuclear Overhauser Effect Difference Spectroscopy By JACOBANGLISTERand FRED NAIDER Introduction Short synthetic peptides have been useful as probes for the antigenic structure of proteins.~ Immunization with a flexible synthetic peptide that is homologous in amino acid sequence with a segment of a protein elicits the production of a spectrum of antibodies. These polyclonal antibodies may recognize different conformations of the peptide and only a fraction of these antibodies may bind the native protein. We have initiated a detailed analysis of the interaction of an immunizing peptide with monoclonal antibodies that either do or do not recognize the native protein. Studies of such complexes should provide insights into antibody function and selectivity. The principal aims of our research are (1) to study the molecular basis of specific antibody-peptide interaction and to understand how the diversity in the amino acid sequence of the antibody affects combining site structure, antibody affinity, and specificity; and (2) to characterize the different antigenic structures of peptide antigens, and to relate peptide conformation to the production of antibodies cross-reactive with the parent protein. H. J. Dyson, R. A. Lerner, and P. E. Wright, Annu. Rev. Biophys. Chem. 17, 305 (1988).
METHODS IN ENZYMOLOGY,VOL. 203
Copyright© 1991by AcademicPrizes,Inc. All rightsof reproductionin any form reserved.
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In order to learn about the molecular bails for the cross-reactivity of antibodies with proteins and peptides derived from them it is necessary to determine the three-dimensional structure of peptide-antibody complexes. Although crystal structures are available for several Fab fragments, only limited success has been achieved on Fab-peptide or Fv-peptide complexes) In principle, nuclear magnetic resonance (NMR) spectroscopy can be used to study the three-dimensional structure of antibody-peptide complexes in solution. Complete structure determination by this technique, however, is currently limited to small proteins of up to 150 amino acid residues. 3-5 Studies on larger proteins are hindered by the loss of spectral resolution, which stems from resonance broadening due to enhanced relaxation pathways and overlap of individual resonances from the increased number of protons. Consequently very few N M R studies of large proteins have been reported. This chapter describes an approach to studying ligand-protein interaction using two-dimensional (2D) transferred nuclear Overhauser effect (NOE) difference spectroscopy. The interaction of antibodies with peptide antigens provides a specific case study. Methodology The antibody molecule has a molecular weight of about 150,000 and contains more than 6000 nonexchangeable protons. To obtain the simplest proton N M R spectrum possible, it is necessary to work with the smallest fragment of the antibody that retains its affinity for the antigen. This fragment is the antibody Fv, which contains about 220 amino acids and has a molecular weight of approximately 25,000. 6 The Fv fragment was used by Dwek and co-workers in their pioneering N M R studies of the antibody MOPC315 complexed with a dinitrophenyl hapten. 7,s Unfortunately the Fv can be obtained by proteolytic cleavage only for a very limited number of antibodies. Therefore, until recently, only the larger Fab fragment was readily available for N M R and crystallographic studies. In addition to the variable domains, the Fab contains one constant domain from each chain. The molecular weight of the Fab is about 50,000. It 2R. L. Stanfield, T. M. Fieser, R. A. Lerner, and I. A. Wilson, Science 242, 712 (1990). 3K. Wfithrich, "NMR of Proteins and NucleicAcids." Wiley,New York, 1986. 4K. WOthrich,Ace. Chem. Res. 22, 36 (1989). 5D. A. Torchia, S. W. Sparks, and A. Bax, Biochemistry 2.8,5509 (1989). 6j. Hochman, D. Inbar, and D. Givol, Biochemistry 12, 1130(1973). 7S. K. Dowerand R. A. Dwek, in "BiologicalApplications of Magnetic Resonance" (R. G. Shulman, ed.), p. 271. AcademicPress, New York, 1979. 8R. A. Dwek, J. C. A. Knott, D. Marsh, A. C. MeLaughlin, E. M. Press, N. C. Price, and A. I. White, Eur. J. Biochem. 53, 25 (1975).
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ANTIBODIES AND ANTIGENS
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contains about 440 amino acids and more than 2000 nonexchangeable protons. Genetic engineering techniques have been used to express the Fv in myeloma cells and Escherichia coli, 9-1~ and it is expected that future NMR studies will take advantage of the increased resolution expected from studies on Fv-antigen complexes (see Wright et al. 12 for a preliminary NMR study on an Fv fragment).
Deuterium Labeling of Antibody The ~H NMR spectrum of the Fab is very poorly resolved even when measured on a 500- or 600-MHz spectrometer. In order to simplify the spectrum and assign resonances of the Fab to specific amino acid types the antibody is labeled with deuterated amino acids.13 This biosynthetic labeling is accomplished by feeding hybridoma cells producing the monoclonal antibody a controlled diet containing selected deuterated amino acids. In studies of antibody-antigen interactions it is beneficial to concentrate on simplifying the spectrum of the aromatic protons for three reasons: (1) that region of the spectrum is a priori less crowded; (2) all aromatic hydrogens can be very efficiently labeled; and (3) aromatic amino acids have a major role in antibody-antigen interactions, with solvent exclusion contributing significantly to the binding energy.14
Two-Dimensional Transferred Nuclear Overhauser Effect Difference Spectroscopy Three-dimensional structure determination by NMR is based on the observation of magnetization transfer between neighboring protons due to the NOE. 15 The magnitude of the NOE is dependent on the reciprocal of the sixth power of the distance between interacting nuclei and is observed up to a distance of approximately 5 A. A two-dimensional (2D) NOE experiment (NOESY) contains the information about magnetization transfer due to the NOE between all pairs of neighboring protons in the molecule.3 Despite the power of 2D NOE spectroscopy this technique has not been applied to the study of the structure of large proteins because of 9 A. Skerra and A. Plueckthun, Science 240, 108 (1988). ~oL. Riechmann, J. Foote, and G. Winter, J. Mol. Biol. 203, 825 (1988). II R. E. Bird, K. D. Hardman, J. W. Jaeobson, S. Johnson, B. M. Kaufman, S.-M. Lee, T. Lee, S. H. Pope, G. S. Riordan, and M. Whitlow, Science242, 423 (1988). 12p. E. Wright, H. J. Dyson, R. A. Lerner, L. Riechmann, and P. Tsang, Biochem. Pharmacol. 40, 83 (1990). 13j. Anglister, T. Frey, and H. M. McConnell, Biochemistry 23, 1138 (1984). ,4 E. A. Padlan, Proteins: Funct. Struct. Genet. 7, 112 (1990). ~5j. H. Noggle and R. E. Sehirmer, "The Nuclear Overhauser Effect: Chemical Applications." Academic Press, New York, 1971.
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the poor spectral resolution found in 2D NMR spectra of these macromolecules. Specific deuteration of aromatic amino acids significantly improves the resolution in the NOESY spectrum; however, cross-peak overlap still prevents resonance identification.~ In order to discern protein-fig,and interactions and the conformation of the bound ligand using the NOESY spectrum of the complex, one must discriminate between cross-peaks due to intermolecular interactions and the numerous cross-peaks due to intramolecular interactions in the complexed protein. Most pertinent is the fact that it is almost impossible to identify the bound ligand resonances, since they are at least as broad as the protein resonances and often considerably broader due to a fast ligand off-rate. Therefore the bound ligand resonances are unresolved from the background. To circumvent this problem we take advantage of a phenomenon known as transferred nuclear Overhauser effect spectroscopy (TRNOE). ~,18 This phenomenon is observed when the ligand molecules exchange rapidly between the bound and the free state. Under conditions where the ligand off-rate is fast relative to the TI relaxation time of both the Fab and the ligand protons, and to the mixing time used in the NOESY experiment, the spectrum of the Fab in the presence of excess ligand (excess spectrum) contains TRNOE cross-peaks. These cross-peaks reflect magnetization transfer that occurs between protons in the bound state. Transfer can be between Fab protons and protons of the bound ligand and/or between different protons of the bound ligand. This transferred magnetization is observed via the free ligand due to the relatively rapid exchange between the bound and free fig,and. These inter- and intramolecular TRNOE cross-peaks are accompanied by numerous cross-peaks due to intramolecular interactions between Fab protons. Since there is essentially no free ligand, none of the TRNOE cross-peaks appears in the NOESY spectrum of the Fab saturated with ligand at a 1 : 1 molar ratio (saturated spectrum). However, the numerous cross-peaks due to intramolecular interactions in the saturated Fab are the same as those in the NOESY spectrum of the Fab in the presence of a ligand excess. By subtracting the saturated spectrum from the excess spectrum, one obtains a difference spectrum in which only TRNOE cross-peaks are observed, t9 This TRNOE difference spectrum is usually well resolved. Its resolution may be further improved by specific deuteration, which simultaneously allows the assignment of cross-peaks to a specific type of amino acid. 16j. Anglister, Q. Rev. Biophys. 23, 173 (1990). ~7P. Balaram, A. A. Bothner-By, and E. Breslow, J. Am. Chem. Soc. 94, 4017 (1972). t8 j. p. Albrand, B. Birdsall, J. Feeney, G. C. K. Roberts, and A. S. V. Burgen, Int. J. Biol. Macromol. 1, 37 (1979). 19j. Anglister, R. Levy, and T. Seherf, Biochemistry 28, 3360 (1989).
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It should be noted that intramolecular NOE in small molecules has a positive sign, with intensity decreasing as a function of molecular weight. For molecules having a molecular weight of about 1500 the NOE is vanishingly small or zero. For larger molecules it becomes negative, and the intensity increases to a maximum o f - 1 with increasing molecular weight. Therefore, in the presence of a binding protein, the sign of transferred NOE cross-peaks due to intramolecular interactions in the bound ligand usually will be negative. The exact dependence of the TRNOE intensity as a function of the binding constant, ligand off-rate, molar ratio between ligand and protein, averaging of the resonances of bound and free ligand, and the cross-relaxation rate in the free ligand is thoroughly described by Clore and Gronenborn. 2°,2~
Assignment of TRNOE Cross-Peaks Cross-peaks appearing in the TRNOE difference spectrum arise from the following: (1) exchange between bound and free ligand, (2) magnetization transfer between antibody protons and free figand protons via the bound state, (3) intramolecular magnetization transfer within the bound ligand via exchange with the free. Therefore at least one of the two frequency values of a TRNOE cross-peak should be of a free ligand proton, while the corresponding second resonance is (1) the same proton of the bound ligand, (2) an antibody proton or (3) another proton of the free ligand. The assignment of cross-peaks to their respective free ligand protons is based on the 2D homonuclear correlated spectrum (COSY) of the ligand, t9 If changes in chemical shift of the ligand protons on binding are large relative to the fig,and off-rate, averaging of bound and free ligand proton resonances is prevented and the COSY spectrum of the ligand can be used. However, for fast off-rates, resulting in averaging of the resonances of the bound and free ligand, one should measure the COSY spectrum of the Fab solution in the presence of the same ligand excess used for the NOESY measurements. Contributions of the Fab to the COSY spectrum mostly vanish due to the linewidth of the Fab protons, and only the contributions of the much smaller ligand are observed. When the resonance of a ligand proton interacting with the antibody falls in a region where a few ligand resonances overlap, unambiguous assignment requires repetition of the experiment with a specifically deuterated ligand. 20 G. M. Clor¢ and A. M. Gronenborn, J. Magn. Reson. 48, 402 (1982). 2~ G. M. Clore and A. M. Gronenborn, J. Magn. Reson. 53, 423 (1983).
[ I 0]
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FIG. 1. Two-dimensional difference spectra between the NOESY spectrum of TE32 Fab with a fourfold excess of the peptide CTP3 (VEVPGSQHIDSQKKA) and the NOESY spectrum of the ~ptide-saturated Fab, showing interactions of specific types of antibody aromatic residues with peptide residues. Assignment to antibody residues is marked by capital letters and arbitrary numbers, while assignment to ~ptide residues is marked by lower-case letters and their location in the sequence. (A) Interactions of antibody tyrosine and laistidine residues with peptide residues. Antibody phenylalanine and tryptophen residues are perdeuterated, while tyrosine residues are deuterated at 2,6-phenyl positions. (B) Interactions of antibody tryptophan and histidinc residues with peptidc residues. Antibody phenylalanine and tyrosine residues are perdeuterated. (C) Interactions of antibody phenylalanine and histidine residues with peptide residues. Antibody tyrosine and tryptophan residues are perdeuterated.
Our studies concentrate on the interactions between aromatic residues of the antibody and the amino acids of a peptide of cholera toxin (CTP3). CTP3 (VEVPGSQHIDSQKKA) 2ta contains only one aromatic residue, histidine, and the resonances of the free peptide histidine can be easily identified. When interaction between a nonaromatic peptide proton and an aromatic proton is observed, and the chemical shifts of the aromatic protons differ from those of the peptide histidine imidazole protons, it is assigned to antibody aromatic residue. The assignment to a specific type of amino acid is based on specific deuteration of the aromatic amino acids of the antibody, t9 Figure 1 shows the assignments of the TRNOE cross-peaks 2~aSingle-letter abbreviations used for amino acids: A, alanine; R, arglnine; N, asparaglne; D, aspartic acid; C, cysteine; Q, glutamine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine.
8.0
234
ANTIBODIES AND ANTIGENS
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to the aromatic residues of the TE32 anti-CTP3 antibody based on the measurements of the 2D TRNOE difference spectrum for three preparations of Fab differing in the labeling of antibody aromatic residues. When one of the two chemical shifts of a cross-peak is of a free ligand proton and the difference between the two is less than about 2 ppm, this cross-peak may be due to exchange. Assignment of such cross-peaks is facilitated by repeating the measurements at different temperatures. At lower temperature the Fab resonances become broader, whereas those of the bound peptide become narrower due to a slower off-rate. Such narrowing can be easily detected if the off-rate is on the order of or faster than the reciprocal of the intrinsic /'2 of the protons in the complex, when both chemical shifts of a cross-peak in the TRNOE difference spectrum are the same as those of the free ligand protons and the line structure of the two resonances involved is observed, this cross-peak is assigned to intramolecular interaction in the bound ligand.
Experiments Involving Slow Ligand Off-Rate When the ligand off-rate is slow relative to the mixing period used in the NOESY experiment and relative to the spin-lattice relaxation time of the Fab and peptide protons, the TRNOE becomes much weaker or nonobservable. We encountered this problem while studying the interactions between the CTP3 peptide and the TE34 antibody.22 In order to be able to use TRNOE difference spectroscopy we searched for minor modifications of the peptide that would decrease the binding constants and enhance its off-rate. We found that conversion of the C-terminal carboxyl into an amide decreases the binding constant of the peptide to TE34 by two orders of magnitude while increasing its off-rate similarly. Indeed, TRNOE difference spectra with excellent signal-to-noise ratio were obtained using the modified peptide. To verify that this modification did not alter the interactions of the antibody with the unmodified portion of the peptide we repeated the experiments with a truncated peptide, acetyI-IDSQKKA, corresponding to residues 9 to 15 of CTP3. This peptide analog lacks one residue (His-8) from the epitope found to be recognized by TE34; however, it contains an intact C-terminal carboxyl. The binding constant of this peptide analog to TE34 is only one order of magnitude less than that for CTP3 binding to the antibody. Our experiments show that both CTP3 with an amide at the carboxyl terminus and the truncated analog exhibit very similar cross-peaks. Thus, we conclude that minor modification of the epitope recognized by the antibody does not significantly affect antibody interaction with the remainder of the epitope. 22 The use of modified peptides that overlap the antigenic epitope permits study of the interac-
[10]
N M R FOR STUDYING PEPTIDE-ANTIBODY COMPLEXES
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FIG. 2. Two-dimensional TRNOE difference spectra obtained after specific chain labeling. (A) Interactions of the light-chain aromatic residues of TE34 Fab with the NCA peptide (Ae-VEVPGSQHIDSQKKA-NH2). (B) Interactions of heavy-chain aromatic residues with the NCA peptide. (C) Interactions of aromatic residues from both chains with the NCA peptide. Assigned antibody residues are marked by capital letters and arbitrary numbers; peptide protons are marked by small letters and numbers denoting the location in sequence of the residues to which the protons belong.
tions between the antibody and the whole peptide. The strategy that we developed can be used to extend the applicability of TRNOE difference spectroscopy to the study of other protein-ligand complexes involving slow ligand off-rates.
Chain Labeling Antibody light and heavy chains can be separated and later recombined to reconstitute the native conformation of the antibody. 23,24This procedure can be used to label each of the chains and subsequently assign resonances to a specific chain. 25 For example, two differently labeled Fab samples (one deuterated, the other not deuterated) are prepared, the interchain disulfide bond is reduced and alkylated, and the chains separated by ion-exchange chromatography under denaturing conditions. The deuterium-labeled heavy-chain fragment is recombined with the unlabeled fight chain and vice versa. The native conformation is then recovered by dialyzing away the denaturant. Figure 2 shows the TRNOE difference spectra obtained for reconstituted Fab in which the chains were specifically labeled.26Figure 2A 22j. Anglister and B. Zilber, Biochemistry 29, 921 (1990). F. Franek and R. S. Nezlin, Folia Microbiol. 8, 128 (1963). H. Metzger and M. Mannik, J. Exp. Med. 120, 765 (1964). 25j. Anglister, T. Frey, and H. M. McConnell, Nature (London) 315, 65 (1985). 26 B. Zilber, T. Scheff, M. Levitt, and J. Anglister, Biochemistry 29, 10032 (1990).
236
ANTIBODIES AND ANTIGENS
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shows the interactions of the aromatic protons of the light chain and Fig. 2B shows the interactions of the aromatic protons of the heavy chain. Figure 2C was obtained for native unlabeled Fab and shows the interactions of aromatic protons from both chains. Figure 2C correspond very well to the sum of the spectra presented in Fig. 2A and B. Assignment of the Antibody Interactions to Specific Residues The assignment of TRNOE cross-peaks allows us to define interactions between various types of amino acids. Given the present state of the art it is impossible to make a complete structure determination of a Fab fragment using NMR spectroscopy. Thus, we are confronted with the problem of extending a given NMR assignment from a type of amino acid to a given residue in the Fab. This problem has been approached using computer model building and conformational energy calculations to generate a starting model for the NMR studies. It was first used by Dwek and his coworkers to study the combining site of an anti-DNP antibody. 7,8 The computerized model building is based on principles introduced by Padlan et aL27 This procedure takes advantage of the homology in the three dimensional structure between various antibodies, which is especially high in the conserved regions that form the immunoglobulin fold. The main obstacle is the modeling of the hypervariable loops, and different approaches to this problem have been developed.28 Using the calculated models, we can judge what amino acids in the combining site are exposed to the solvent and, therefore, have the potential to interact with the antigen. By combining the NMR information, the amino acid sequence data of the six CDRs and the preliminary model, most ligand-protein interactions can be assigned to specific antibody residues. Additional information about interresidue interactions in the antibody combining site can facilitate the assignment of the interactions to the specific residues. Such information can be obtained by calculating the difference between the excess spectrum and the NOESY spectrum of the Fab itself. This difference spectrum contains cross-peaks due to interactions with the antigen and those due to intra-Fab interactions of antibody protons that change their chemical shift on antigen binding.
27 E. A. Padlan, D. R. Davies, I. Pecht, D. Givol, and C. Wright, Cold Spring Harbor Syrup. Quant. Biol. 41, 627 (1976). 2s C. Chothia, A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip, P. M. Coleman, S. Spinelli, P. M. Alzari, and R. J. Poljak, Nature (London) 342, 877 0989).
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Derivation of Constraints on Proton-Proton Distances The 2D TRNOE difference spectrum contains cross-peaks due to all the short-range interactions (proton-proton distance < 4 ~,) between the antibody and the peptide. Measurements of the intensity ofintramolecular magnetization transfer between a pair of neighboring protons after a short irradiation period of one of them or after a short mixing time in a NOESY experiment are used to evaluate the distance between the protons. Such measurements have been carded out to obtain constraints on protonproton distances that were subsequently used to calculate the threedimensional structure of small proteins. 3 Clore and Gronenborn extended the application of such measurements to transferred NOE in proteinligand complexes. 20,2~ Their analysis distinguishes three regions of chemical exchange. In the case of fast exchange between bound and free ligand, when the ligand off-rate is faster then 10 times the spin-lattice relaxation time of its protons and when the resonances of the bound and free ligand are averaged, the magnitude of the transferred NOE (Nn~) between an antibody proton, A, and a peptide ligand proton, H, is given by Nn.A (1 -- a)an~ rm, where trm,~is the cross-relaxation rate between an antibody proton and neighboring bound peptide proton, a is the mole fraction of the free peptide, and rm is the mixing time in the NOESY experiment or the irradiation time in a 1D NOE experiment. This simple relationship applies to both 1D and 2D TRNOE experiments. The distance ratios between different pairs of protons in the complex can be determined from trrtA, which is inversely proportional to the sixth power of the distance between the protons. When the chemical shifts of the bound and free peptide resonances are not averaged but the off-rate is still faster than T1 and ana, and the peptide is present in large molar excess, the TRNOE intensity between an antibody proton and a free peptide proton is still approximated by the above equation. In the case of medium off-rate the efficiency of the TRNOE will depend on the off-rate 2°,21 and the above equation for Nn,~ can serve only as an upper limit. If the exchange between bound and free ligand is slow on the cross-relaxation and spin-lattice relaxation scale the transferred NOE intensity is vanishingly small. To translate cross-peak intensities into restraints on proton-proton distances one needs a calibration usually obtained by measuring the intensity of a cross-peak between a pair of protons separated by a known and fixed distance. To obtain such calibration we measured the difference spectrum between the NOESY spectrum of TE34 Fab in the presence of 10-fold peptide excess and the NOESY spectrum of the uncomplexed Fab (without spectrum). In addition to cross-peaks due to transferred NOE this
238
ANTIBODIES AND ANTIGENS
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type of difference spectrum shows cross-peaks due to intra-Fab interactions of protons experiencing changes in chemical shift on antigen binding. When all tryptophen and phenylalanine residues ol~ the antibody were deuterated while tyrosine residues were unlabeled the difference spectrum measured for the Fab contains very well-resolved cross-peaks due to intmresidue interactions between the ~ and Jl and between the ~2 and J2 tyrosine protons. These cross-peaks are not observed when the C6 protons of the antibody tyrosine residues are deuterated. The intensity of the resolved cross-peaks is then compared to the intensity of the exchange cross-peak of Ca H of the peptide histidine. Since the exchange cross-peak appears in all the TRNOE difference spectra, and all spectra are measured at the same mixing time and with the same molar ratio of peptide, this cross-peak can serve as an internal standard for calibrating the intensities in all TRNOE difference spectra. Through this internal standard one gets the ratio between cross-peak intensities in the TRNOE difference spectrum and the cross-peak due to intratyrosine interactions and subsequently the ratio between the distances of the corresponding pairs of protons. This analysis indicates that all the cross-peaks due to intermolecular interactions observed in the TRNOE difference spectra are due to strong interactions between protons that are less than 3.5 A apart.
Experimental
Procedures
Sample Preparation Hybridoma cells producing antibodies are grown as monolayers in 700 ml T flasks on RPMI 1640 medium29 supplemented with only 2% fetal calf serum to increase the efficiency of the labeling. Supernatant is collected every 2 - 3 days and 50 ml of fresh medium is supplied to each flask. Antibody production is dependent on the specific cell line and varies between 20 and 60 mg/liter. Biosynthetic specific deuteration of the antibody is accomplished by feeding the hybridoma cells producing the antibodies medium containing the selected deuterated amino acids according to the RPMI recipe. Antibodies are purified utilizing protein A-Sepharose CL-4B column chromatography)°,3t Antibody solution is concentrated by an Mr 25K cutoffcollodion bag (Schleicher & Schuell, Dassel, Germany) to ZgG. E. Moore, R. E. Gerne, and H. A. Franklin, J. Am. Med. Assoc. 199, 519 (1967). GIBCO Laboratories, Grand Island, New York, Technical Manual, 1982. ~oG. Otting, H. Widmer, G. Wagner, and K. WOthrich, J. Magn. Resort. 66, 187 (1986). 3~V. T. Oi and L. A. Herzenberg, in "Selected Methods in Cellular Immunology" (B. B. Mishell and S. M. Shiigi, eds.), p. 351. Freeman, San Francisco California, 1980.
[ 10]
NMR FOR STUDYINGPEPTIDE-ANTIBODYCOMPLEXES
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a concentration of 30 mg/ml. Fab is obtained by papain digestion32 and purified by applying the reaction mixture to a Sephadex G-100 column linked in series to a protein A column (50 ml protein A-Sepharose CL-4B/ 200 mg of antibody). The columns are preequilibrated and developed with 50 m M Tris-HC1, pH 9, containing 0.15 M sodium chloride and 0.05% (w/v) sodium azide. The large peak of Fab is preceded by a much smaller peak of (Fab')2. The Fc binds to the protein A and is eluted with citrate buffer, pH 4.5. The Fab is concentrated to 3 m M with a collodion bag and the aqueous solution is dialyzed against four changes of 10 m M phosphate-buffered dueterium oxide, pH 7.1, containing 0.05% sodium azide. A typical NMR sample contains 500/tl of 3 m M Fab. Antibodies are never lyophilized or frozen. NMR Measurements
NOESY spectra are measured on a 500-MHz spectrometer in the phase-sensitive mode using a proton-selective probe. A mixing time of 100 msec, which was found to be optimal for obtaining maximum intensities for the strong cross-peaks observed, is used. The carrier frequency is set on the HDO line, and a spectral width of 6000 Hz is used. The HDO line is presaturated, using minimal power, for 3 see before the first 90* pulse is applied. Spectra are Fourier transformed in both dimensions after application of a squared cosine window function. A considerable reduction in tl ridges is achieved by using the method of Otting et aL) ° already incorporated in the standard 2D Fourier transformation in Bruker's software. Spectra are not symmetrized. The two spectra used in the difference calculation are measured consecutively, Fab concentration is between 2 and 3.5 m M in 0.01 M phosphate-buffered 1:)2O, pD 7.15, and the temperature is 42 °. Spectra are recorded with 256 values of q ; for each value, 64 or 80 scans are collected that are preceded by 4 dummy scans. Measurements of the TRNOE difference spectra for 60- and 100-msec mixing times exclude the possibility that the cross-peaks observed in the TRNOE difference spectra at 100 msec are due to spin diffusion. Difference Spectra Calculations
The phases of the initial measurements (first tl value) of both 2D experiments are matched to obtain a 1D difference with minimal distortion to resonances and baseline. This is accomplished first by visual comparison of the two spectra and then by fine numerical phase correction of 32 R. R. Porter, Biochem. J. 73, 119 (1959).
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ANTIBODIES A N D ANTIGENS
[10]
one to match the other. The phase corrections thus obtained are applied to the preliminary 2D Fourier transformation. From the NOESY spectrum of the Fab with excess peptide, we select a row crossing a free peptide resonance. The same row is selected from the NOESY spectrum of the peptide-saturated Fab, and the two are subtracted. The phase of the resulting 1D difference spectrum is numerically corrected to obtain a pure LorentzJan for the resonance of the peptide proton. This phase correction is now used to reprocess the two NOESY spectra, and the difference between these rows is reexamined to verify that the resonance of the free peptide retains a pure Lorentzian shape. This procedure ensures the matching of phases between the two 2D spectra in the region containing cross-peaks between nonaromatic protons. The rows and columns crossing the diagonal at the resonances of the histidine imidazole protons of the peptide require additional slight phase correction. Baseline distortions due to t2 ridges are corrected for by subtracting from the 2D difference spectrum the value for a column with no signal at the intersection with the diagonal of the 2D spectrum. Further baseline correction of individual rows is carried out where necessary by fitting a baseline with a fourth-order polynomial.
Conclusions and Perspectives The methodology developed in our laboratory allows one to obtain detailed information concerning the interactions between residues in a peptide antigen and residues in the combining site of a monoclonal antibody. Transferred NOE difference spectroscopy, in conjunction with specific deuteration of the antibody, provides parameters that give information about distances between the above residues. Unfortunately, the number of NOE cross-peaks are not sufficient to calculate an unequivocal structure for the bound antigen. To circumvent this problem we apply models for the antibody combining site that are predicted based on structures for CDRs determined by X-ray crystallography. These models help us to verify assignment of the peptide-antibody interactions and can be used along with NOE distance constraints, energy minimization, and molecular dynamics calculations to provide a 3D model of the structure of the bound peptide antigen. Moreover, comparison of NMR data with calculated models allows one to examine the validity of a given model and to exclude "bad models" that result from choosing improper segments from the known three-dimensional structures of other antibodies. It is important to emphasize that the above approaches are warranted by the severe overlap problem that is confronted in studies of the interaction of peptides with large proteins. This problem has been encountered by others and elegant
[ 11 ]
ISOTOPE-EDITEDNMR
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procedures involving isotope editing 33,34 and 2D difference spectroscopy with deuterated ligands have been developed. 35 In all cases to date, information forthcoming from such studies requires comparison with crystal structures to rationalize interactions that are apparent in the N M R spectra. The combined application o f 2D T R N O E difference spectroscopy and isotope editing procedures holds great promise for unraveling the mysteries o f p e p t i d e - p r o t e i n interactions. At present, despite some obvious shortcomings, these are the only approaches to study the conformation o f peptides b o u n d to antibodies, enzymes, or receptors in solution. Acknowledgments We are most grateful to Michael Levitt, Rina Levy, Taft Scherf, and Barbara Zilber for their important contributions to the work describedin this chapter. This work was supported by the United States-Israel Binationfl ScienceFoundation. F. N. is a visiting professoron a Fulbright International ExchangeProgram. 33S. W. Fesik, J. R. Luly, J. W. Erickson, and C. Abad-Zapatero, Biochemistry 27, 8297 (1988). P. Tsang, T. M. Feiser, J. M. Ostresh, R. A. Houghten, R. A. Lerner, and P. E. Wright, Fronteirs NMR Mol. Boil., 63 (1990). 32S. W. Fesik and E. R. P. Zuiderweg,J. Am. Chem. Soc. 111,5013, (1989).
[11] Isotope-Edited Nuclear Magnetic Resonance Studies of F a b - P e p t i d e Complexes
By P. TSAN6,M. RANCE,and P. E. WRmHT Introduction Through the development o f isotope-edited techniques, 1-3 the application o f nuclear magnetic resonance ( N M R ) spectroscopy to studies o f higher molecular weight systems such as F a b - a n t i g e n or F a b ' - a n t i g e n complexes has become feasible. 4-7 The general concept and principles o f this m e t h o d will be described briefly in this chapter, and illustrated with results obtained in our laboratory. R. H. Griffeyand A. G. Redfield, Q. Rev. Biophys. 19, 51 (1987). 2A. Bax, S. W. Sparks, and D. A. Torchia, this series, Vol. 176, p. 134. 3G. Wagner, this series, Vol. 176, p. 93. 4 p. Tsang, T. M. Fieser, J. M. Ostresh, R. A. Lerner, and P. E. Wright, Pept. Res. 1, 87 (1988).
METHODS IN ENZYMOLOGY, VOL. 203
~ t © 1991 by Academic Press, Inc. All righls o f ~ l ~ d u ~ o n i n any form reserved.
