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Antibody-antigen interactions: new structures and new conformational changes
Antibody-antigen interactions: new structures and new conformational changes lan A Wilson and Robyn L Stanfield The Scripps Research Institute, La Jolla, USA During the past year, many new antibody structures have been determined, increasing our understanding of these immunologically important molecules. Of special interest are new catalytic antibodies, antibody-peptide and antibody-virus complexes, NMR structures, and structures illustrating conformational changes and antibody cross-reactivity. Current Opinion in Structural Biology 1994, 4:857-867 Introduction Table 1. Fab and Fv X-ray structures reported during the review period.
During the past year, 23 reports o f new antibody or antibody binding fragment (Fab) structures have appeared (Table 1), bringing the current total o f such structures to well over 50; about 40 of these are currently deposited in the Brookhaven Protein Data Base [1]. The growing number o f structures has led to more statistically valid conclusions about the nature o f antibody-antigen recognition, while continuing to provide some new surprises about features o f this interaction. We now have a clearer understanding o f the atomic details o f antigen binding to antibody, as well as of the role and extent o f conformational changes in this molecular recognition event. The accumulation o f structures is also useful for the prediction and determination of new antibody structures, for use in chemistry, biology, and medicine. Perhaps the most interesting new results that emerged during the review period were the first glimpses o f the three-dimensional architecture of three catalytic antibodies. Whereas natural enzymes have had a very long time to evolve their catalytic function, antibodies that catalyze chemical reactions (abzymes) can be produced in the laboratory in only a few weeks. While the first abzymes catalyzed only simple hydrolytic reactions, the present generation can carry out transformations that are disfavored or for which no natural enzyme catalyst is known [2-5]. Two o f the recently determined abzyme structures illustrate the different mechanisms they use in their catalysis [6",7"°]. Other major advances reported during this period concern the nature and role of conformational changes in antibody-antigen recognition. It is quite clear now that there is a large variation in the extent to which conformational changes occur in individual antibodies. Undoubtedly, further examples that extend the range and size o f these changes will be discovered [8°]. Still at issue is whether one can describe a universal mechanism for antibody-antigen recognition. The
Fab
CNJ206
Antigen
p-nitrophenyl
Unliganded
Liganded
Resolution
Reference
+
-
3.0
[29]
ester 17E8
Norleucine
-
+
2.5
[7 ° ° ]
1F7
phosphonate Transition state
-
+
3.0
[6 e°]
58.2
analogue gp120
-
+
3.0
[32 eel
peptide 17/9
Hmg peptide
+
+
2.8
[15 e]
TE33
CTP3 peptide HRV2 peptide PHL FIPV antibody
-
+
2.3
[31]
+
+
2.5
[17 ° ° ]
-
+ +
2.4 2.9
[70] [71 e]
+
+
2.7
[1, 5, 13,
-
+
2.2, 2.8
[54]
8F5 Dl1.15 409.5.3 DB3 CHA255
Steroid haptens Metal chelate
Je172
haptens DNA
+
2.7
[81]
Je1318 NC6.8
DNA Sweetening
+ +
+
2.8 2.6, 2.2
[82] [16]
50.1
compound gp120
+
+
2.8
[8#,12]
peptide D1.3 Fv
HEL
+
+
1.8
[50e]
D1.3
HEL
-
+
1.8
[68]
mutant 26-10
Digoxin
-
+
2.7, 2.5
[49]
N C l 0 scFv NC10
Neuraminidase Neuraminidase
-
+ +
3.0 2.5
[76] [53 e°] [46]
17-1A
HRVl 4
+
-
2.7
HuH52-OZ
CDI 8
+
-
3.0
[75]
HuH52-AA Fv
CD18
+
-
1.9
[75]
Hmg, hemagglutinin; PHL, pheasant egg lysozyme; FIPV, feline infectious
peritonitus virus; HEL, hen egg lysozyme.
current trend favors induced fit [9-13], and some new examples [8°,14°',15°,16,17 °°] add support to that hy-
Abbreviations
Ab2--anti-idiotypic antibody; Fab---antibody binding fragment; Fv--Fab variable domain. © Current Biology Ltd ISSN 0959-440X
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Proteins pothesis. These examples still do not constitute proof, however, as the X-ray structures represent single snapshots of a dynamic situation. High-resolution N M R studies, combined with detailed kinetic and thermodynamic measurements and molecular dynamic simulations, are now providing welcome new information to help resolve these issues. In the end, it may turn out that antibody-antigen recognition cannot be explained by a simple model, such as for peptide antigen recognition by MHC class I [18-20] and M H C class II [21,22] molecules. Undoubtedly, the most controversial paper in this period [16] revisits the question of whether antigen binding causes a signal to be transmitted from the antibody combining site to the more distal constant domains, thereby initiating the effector response that includes complement and Fc receptor binding. For such a complicated problem, both the uncleaved, unliganded antibodies and their complexes probably need to be crystallized and studied. The recently determined X-ray structure of an intact IgG that contains a normal, rather than deleted, hinge region shows there is still some promise for this approach [23].
Catalytic antibodies Several years after the initial demonstration that chemical reactions can be catalyzed by antibodies produced
through immunization of mice with analogues of the transition state of the reaction [24-27], a flurry of activity has resulted in the structural determinations of several abzymes. The crystal structure of an antibody (1F7) that catalyzes a chorismate mutase transformation shows that its endo-oxabicyclic dicarboxylic acid transition state analogue (TSA) is bound in a small but highly complementary depression on the antibody surface [6"] (Fig. la). The antibody does not appear to require any functional groups to participate in the catalysis, and therefore seems to work mainly by restricting the substrate to a conformation that mimics its higher energy, pericychc transition state. A second abzyme crystal structure [7"'] (Fig. lb) supports the notion that catalytic antibodies may mimic natural enzymes by containing arrangements of catalytic groups that resemble those found in known enzyme structures. On the basis of the X-ray structure of 17E8 (an active hydrolytic antibody) in combination with kinetic data, Zhou et al. [7°°] propose that the mechanism of hydrolysis by the antibody is similar to that found in serine proteases. Indeed, the antibody combining site contains two residues from the heavy chain (His H35 and SetH95; numbering as in [28]) that appear to be topologically similar to the equivalent components of the Ser-His-Asp catalytic triad in serine proteases. AdditionaUy, the residue LysH93 from the antibody serves as the 'oxyanion hole'. In the crystal structure, the hydroxyl group of Set H95 is rather far (5-6 A) from the phospho-
(a)
(b)
Fig. 1. (a; top) Stereoview of the catalytic Fab 1F7. The binding site for 1F7 is shown with the bound transition state analogue. The light chain is on the left. (b; bottom) Catalytic Fab 17E8. The hydrolytic Fab with antigen is in the same orientation as 1F7 above. All figures created with MOLSCRIPT [83].
Antibody-antigen interactions Wilson and Stanfield rus atom of the hapten and makes a hydrogen bond with a neighboring TyrH97. Zhou et al. [7°°] show that rotations of both the serine and histidine sidechains can put them into position to carry out their proposed function. A structure determination of 17E8 at the pH optimum of the enzymatic reaction (around pH 9.0) is currently underway to test this hypothesis (TS Scanlan, personal communication). It is also interesting to look at the frequency of histidine and serine at these positions in the variable region of IgG heavy chains. Histidine is the residue found most frequently at position H35 (in 378/1459 protein sequences studied), whereas aspartate is the' residue found most frequently at position H95 (in 289/1451 cases); serine is found at position H95 less frequently, in 187 of 1451 sequences studied [28]. A third structure of an unliganded Fab with esterolytic activity (CNJ206) [29] reveals the binding site for the para-nitrophenyl ester substrate. On the basis of a structural model for the complex with the transition state analogue, the authors [29] propose that catalysis also involves an oxyanion hole similar to that found in serine protease active sites (and in Fab 17E8). Whether such details of ligand binding can be modeled from the unliganded structure obviously awaits the X-ray structure determination of the ligand-bound form of the antibody.
Antibody-peptide complexes The feature most commonly seen in antibody-peptide complex structures is a ~-turn in the peptide that is deeply embedded in the antibody-antigen interface. Several examples of this recognition motif now exist and inClude a type II [~-turn (VPHK, residues 71-74; singleletter amino acid code) in a peptide from myohemerythrin [10], a type I [~-turn (DYAS, residues 104-107) in an influenza hemagglutinin peptide [12], and two tight turns in the peptide hormone angiotensin II [30]. Three new examples from this review period are a type II ~turn in the cholera toxin peptide CTP3 bound to Fab TE33, (VPGS, residues 52-55; [31]), a type I ~-turn in the VP2 peptide from human rhinovirus 2 (HR.V2) bound to Fab 8F5 (AETP,.,2158-2161; [17°°]) and a type II ~-turn in a peptide from human immunodeficiency virus 1 (HIV-1) gpl20 bound to Fab 59.1 (GPGk, residues 319-322; [32°°]). These last two examples actually contain more complicated turn arrangements than those mentioned above. In the 8F5-VP2 complex, the peptide (VKAETRLNPDLQPTE-NH2, residues 2156-2170) contains an asparagine-pseudoturn (NPD) following the [~-turn. The HIV-1 peptide in the 59.1 complex has an unexpected double turn, where the predicted type II ~-turn [33] is followed by a combined type III (GR_AF)- type I (RAFY) turn. In all these structures, anywhere from 7 to 12 of the peptide residues are ordered, no matter how long the peptide in the crystal (up to 24 residues in the 59.1 complex). Thus, the antibody interactions must extend beyond the [3-turns, and include extended ~-strand type interactions between the peptide and antibody in
the 17/9-hemagglutinin [12,15°], TE33-cholera toxin [31] and 8F5-VP2 complexes [17°']. The only example that does not involve interaction with a tight turn is the Fab 50.1-HIV-1 peptide complex, in which the peptide is essentially extended and runs along a 25 ]x groove between the variable light and heavy domains (VL and VH) [34]. Even in this structure, however the carboxyterminal end of the peptide starts to adopt a turn-like structure before exiting the binding site. In the cholera toxin peptide paper [31], Shoham suggests that "antipeptide antibodies seem to induce a ~turn conformation in the bound peptide irrespective of the sequence of the peptide" and cites as examples the B1312 and 17/9 structures [10,12]. Secondary structure predictions for these peptide sequences, however, show that they have a marked tendency to form the turn types found in the Fab-peptide structures, as in the recent examples of the G P G R sequence (predicted to be type II) in the HIV-1 peptide and VPGS (predicted to be type II) in the cholera toxin CTP3 peptide. Therefore, it is not likely that the antibody randomly induces peptides to adopt ~-turns. O f more relevance is how the bound peptide structures correspond to their cognate structures in their respective proteins. The myohemerythrin (B1312) and cholera toxin (TE33) anti-peptide antibodies do not bind to the native versions of the proteins, except in solid-phase enzyme-linked immunosorbent assay (ELISA) systems (TE33; [31,35]). However, they may react with nonnative forms, such as the binding of apomyohemerythrin by B1312 [10]. The myohemerythrin peptide sequence in holomyohemerythrin is largely helical [36], whereas the peptide sequence in the intact cholera toxin B-subunit [37] has a type I ~-turn at residues Gly-SerGlu-His (positions 54-57) and an or-helix from residues 59-64. On the other hand, three anti-viral antibodies (50.1 and 59.1, against HIV-1 gp120), and 8F5, against (I-IRV2 VP2) neutralize viral infectivity and presumably react with native protein. The anti-influenza antibody 17/9 and several related antibodies bind to the biologically important fusion form of influenza virus hemagglutinin at low pH [38]; the rhinovirus antibody was raised against native virions [17 °°] and the HIV-1 gpl20 antibodies neutralize and inhibit various viral functions [32°°]. Hence, the influenza hemagglutinin, rhinovirus VP2, and gpl20 peptides would be expected to mimic their protein counterparts [12,15 °, 17°°,34]. An extensive study of various hemagglutinin peptides in complex with Fabs 17/9 [15 °] and 26/9 [39] shows that the only major difference between the antibody-bound peptide conformation and its counterpart in the intact hemagglutinin is in the ~ angle of residue Prol03. Two sequence changes in the binding site between the closely related 26/9 and 17/9 antibodies leave the overall bound peptide conformation virtually unchanged except for an additional interaction with a water molecule [39]. Although the structure for the 8F5 antigen, HRV2, has not been determined, the related HRV1 structure contains a loop that, despite its difference in sequence, is very similar to the corresponding HP,.V2 peptide structure, except for a
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Proteins cis-proline (KAETRLNPDLQ). The structure for HIV1 gp120 is not yet known, but an N M R structure o f the 40 amino acid loop containing the sequence of the peptides bound to Fabs 50.1 and 59.1 also indicates that the G P G R sequence tends to form a a-turn [40]. Thus, it is clear that antibodies generated against peptides or peptide analogues (17/9, 26/9, 50.1, 59.1), or the intact virion (8F5), can cross-react with both peptide and protein and hence can provide valuable information for vaccine development or drug design [32°°]. Indeed, the structures o f the two Fab complexes o f 50.1 and 59.1 with different but overlapping peptides o f the V3 loop of HIV-1 gp120 have enabled a conformation for the top of the V3 loop to be assembled by stitching together the structures of the two peptide pieces. A more comprehensive recent analysis of peptide-antibody complex structures can be found elsewhere [41].
Virus-antibody structures A combination of electron microscopy and X-ray diffraction is now being used to elucidate the details of how neutralizing antibodies bind to viruses. A pioneering study on a cowpea mosaic virus with an Fab fragment of a neutralizing antibody, using cryo-electron microscopy and image reconstruction at 23 fi~ [42], showed the footprint of the Fab on the viral capsid protein. A recent study [43] with the intact IgG showed monodentate binding to the cowpea mosaic virus, which contrasts with the bidentate binding to rhinovirus 14 (HRV14) by the antibody 17-IA [44",45]. The Fab and the whole IgG o f l 7 - I A both bind across the icosahedral twofold around the fivefold axes; an X-ray structure o f the Fab fragment of this antibody [46] has now enabled a better fit to be made for the image reconstruction. It suggests that the virus itself does not change its gross conformation on binding of Fab or IgG [44°,45]. More details of how intact viruses are recognized by monoclonal antibodies are likely to come soon from the combination of X-ray diffraction and electron microscopy.
Conformational changes Several types, of conformational rearrangements have been documented in Fab fragments in response to ligand binding. In our previous review in Current Opinion in Structural Biology [47], the changes outlined included small side-chain rearrangements, segmental movements of complementarity determining region loops (CDRs; L1, L2, L3 and H1, H2, H3 from the light and heavy chain respectively), large rearrangements o f C D R H3 and alterations in the relative disposition o f the VL-V H domains. The Fabs 17/9 [12] and BV04-01 [11] exhibited the largest of these changes in CDtk H3 (3-5 ,~ in backbone atoms for 17/9) and in relative VL-V H rotation (7.5 ° for BV04-01). An even larger relative rotation (16 °) of the VL-V H domains has now been observed for the anti-HIV-1 Fab 50.1 on complex formation with the V3 loop peptide [8°] which results in a significantly altered combining site in the peptide-bound Fab. Super-
imposed on this quaternary structural change is the now commonly seen rearrangement o f the H3 loop, which moves out o f the binding site with root mean square deviations o f up to 5 A in backbone atoms and 6A in side-chain atoms compared to the unliganded Fab. A detailed analysis o f the somewhat limited database containing both unliganded and liganded Fabs indicates that the larger domain rotations are associated, perhaps not surprisingly, with smaller buried surface areas (1000A2) between the VL and VH domains, compared to smaller domain rotations (0°-4 ° rotations) that have VL-V H buried surface areas of up to 1700 A2 [8"]. Thus, a smaller VL-V H interface may be more likely to be subject to such quaternary rearrangements on ligand association. These results constitute experimental proof of the interface adaptor hypothesis put forward by Colman [48], who proposed that when the size of the antigen interface approaches the size of the VL-V H interdomain interface, then VL-V H rearrangements might accompany and play a role in antibody-antigen complementarity. Other examples o f unliganded and liganded structures reported during this review period include the antisweetener Fab NC6.8 [16], the anti-rhinovirus Fab 8F5 [17"'], the anti-digoxin antibody 26-10 [49], the highresolution refinement o f the anti-lysozyme Fv (variable domain o f the Fab) D1.3 [50], and the steroid binding Fab DB3 [14"°,51°']. These Fabs and Fvs show varying VL-Vkl rotations upon ligand association: 2.8 ° (NC6.8, 1295A2 VL--VH interface size); 0 ° (DB3, 1425fi~2); 2 ° (26-10, 1637fi~2); and 2.2 ° (D1.3 Fv, 1469fi~2). Thus at present, the majority OfVL-V H rearrangements are clustered around 2-6 °, with only BV04-01 and 50.1 showing larger rotations. A review of the impact of these changes on structure prediction and design of antibodies has been discussed elsewhere [52]. Fabs are also very flexible at the junction between the variable (V) and the constant (C) superdomains; in one recent Fab structure (NC10), only the Fv (VL-VH) is ordered in the crystal [53"']. The so-called elbow angle is a measure o f the relative disposition of these superdomains and corresponds to the angle between the two pseudo twofold rotation axes of the V and C superdomains (Fig. 2). The range o f elbow angles increased during the review period and now varies from 127 ° for unliganded Fab 8F5 [17"] to 194 ° for Fab CHA255 [54] (Table 2), although a set o f unpublished coordinates which have been deposited in the Brookhaven Protein Data Bank for Fab HIL (pdb8fab.ent; FA Saul, tkJ Poljak, depositors) has an elbow angle of 227 ° for one of the two unliganded molecules in the HIL structure. There are now several examples of two Fabs in the same crystallographic asymmetric unit having different elbow angles, both for unliganded Fabs (2130 and 227 ° for HIL [pdb8fab.ent] and 159 ° and 167 ° for Je142 [55]) and for liganded Fabs (164 ° and 177 ° for 50.1 [34]). Differences in elbow angles have also been reported between unliganded and liganded structures of the same Fab (161 ° and 176 ° for 17/9 [12], 176 ° and 164 ° for 50.1 [8°], and 190 ° and 153 ° for NC6.8 [16]). The last example, the anti-sweetener Fab, NC6.8, prompted Guddat et
Antibody-antigen interactions Wilson and Stanfield al. [16] to revisit an old theory about signal transmission
from the variable to the constant domain upon hgand binding. This idea was popular in the mid-1970s [56], when a closed or bent elbow angle was thought to be characteristic of antigen-bound antibodies and an open or straight elbow angle (approaching 180 °) characteristic of unliganded antibodies. Not until the solution of the first Fab-protein complex structure (D1.3-1ysozyme [57]) was this suggestion disproved [58]; however, Huber did not completely rule out the idea of "elbow bending as a signal transfer mechanism" [58], which had also been suggested from light chain dimer studies [59]. Table 2. Elbow angles. Unliganded Fab
PDBa code
Angle (0) Mol 1 Mol 2
Liganded Fab
PDB code
Angle (°) Mol 1 Mol 2
8F5* NEW* McPC603* B72.3" J539" HC19" YST9.1* hu4DS-ver4* hu4DS-ver7* KOL* 36-71" 3D6" R19.9" HIL*
lbbd 7fab 2mcp lbbj 2fbj 1gig 1roam lfvd 1fve 2fb4 6lab ldtb lfai 8lab
127.3 130.2 133.3 137.6 143.9 146,7 150.9 156.9 155,3 1 5 7 . 1 154.8 166.9 167.0 176.1 t76,8 212.9 226.9
1F7 59.1 TE33 HyHEL10 NC41 NC4i NC41 NC41 AN02 HyHEL-5 HyHEL-5 D1.3 26/9 Se155-4 Se155-4 Se155-4 Set55-4 4-4-20 CHA255 CHA255
1fig lacy ltet 3hfm lnca tncb lncc lncd 1baf 2iff 2hfl lfdl lfrg lmfe 1mfb 1mfd lmfc 4lab lind line
132.3 134.8 141.2 146.4 147.1 t47.4 147.9 147.9 154.2 16t.9 162.5 173.8 175.3 183.9 184.0 184.0 184.8 186.0 194.2 194.3
B1312 Je142 17/9 17/9 17/9 50.1 50.1 DB3 D83 DB3 DB3 DB3 26-10 BV04-01 NC6.8
2igf I jel lhim thin lifh Iggi Iggi Idbb Idbj Idbk ldbm 2dbt ] igj lcbv 2cgr
157.4 154.9 174,2 177.3 175,9 163.9 163,9 182.6 182.5 182.6 182.5 182.3 183.9 175.5 152.9
81312 Je142 17/9 17/9 17/9 50.I 50.1 DB3 DB3 DB3 DB3 DB3 26-10 BV04-01 NC6.8
ligf b lhil lhil lhil Iggb Iggc Idba Idba Idba ldba tdba 1igi lnbv 1cgs
154.3 159 161.2 161.2 161.2 173.5 175.6 181.7 181.7 181.7 181.7 181.7 186.2 188.2 189.8
156.6 167 161.8 161,8 161.8
173.1
176.5 176.5
184.0
apdb code indicates the protein databank entry information. Unliganded Fab elbow angles for which corresponding liganded Fab elbow angles have not been determined are asterisked (*). bNot deposited in the protein data bank,
Currently, such internal variations in elbow angles have been documented only with Fab structures, although a cryo-electron microscopy analysis of the neutralizing antibody 17-IA bound to human rhinovirus 14 suggests that the bidentate IgG has an elbow angle different from the Fab fragment alone [44°]. Thus, it is somewhat surprising that the NC6.8 structure mentioned above has been used to invoke the transmission theory, given that only one of the ligand complexes, a trisubstituted guani-
dine hapten bound to NC6.8, crystallizes with a different elbow angle, whereas two others appear to resemble the unliganded Fab NC6.8. By the authors own calculations, other structures, such as Fab 17/9, do not conform to their more detailed concerted shifts ideas. Clearly, more studies with intact immunoglobulins are needed to resolve such questions of transmitted conformational changes. Indeed, the ligand-induced alterations in VL-V H domain association would seem to be strong candidates for such a signal, but, wisely, no groups have (yet!) come to that conclusion. Further confirmation of flexibility elsewhere in immunoglobnlin molecules was provided by a recent [13C]NMR. study of a murine intact monoclonal IgG2a [60] and by the cryo-electron microscopy study of intact IgGs bound to human rhinovirus 14 [44",45]. In the N M R study, the hinge region connecting CH1 to CH2 was described as a mosaic structure of three components: the upper hinge, a structure including the linked disulfides, and the lower hinge. In the rhinovirus-IgG complex structure, only the Fab fragments could be seen in the image reconstruction for the 30 IgGs that straddle the icosahedral twofold axis of the virus and bind to the VP1 subunits that surround the fivefold axes. These studies confirm the flexibility of the hinge region that was observed early on in the intact Kol structure, in which the Fc region was disordered [56]. Since then, the only intact antibody structures to have been determined are those with deleted hinge regions, such as Dob [61] and Mcg [62], and for an IgG with a normal hinge that surprisingly contained an ordered Fc region [23]. Questions about the number of conformational states that an antibody can adopt were answered in part with the structure determination of five complexes of anti-progesterone Fab' DB3 [14°',51 ''] with different steroids. Two classes of steroid ligands that differ substantially in their shapes were found to bind in alternative orientations in the antibody combining site. The Fab' made minor adjustments in its conformation (0.35 A root mean square deviation for backbone atoms among the steroid-bound forms) in order to accommodate these differently shaped ligands. N M R studies X-ray structures have suggested the existence of distinct unhganded and hganded forms of Fabs [47,52], with major conformational variation occuring in the C D R H3 loop. There are no well documented examples of the other C D R loops adopting substantially different structures or varying from the canonical forms [63] when going from their unliganded to liganded forms. Some X-ray evidence suggests that the C D R L1 can be more flexible at its top [64]; otherwise, there are no obvious trends in decreased mobility in the CDl
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Fig. 2. Elbow angles. In this stere0view, several Fabs have been superimposed by their variable domains, so the differences in their elbow angles are visible. The current range of elbow angles ranges from 127°for 8F5 (1 bbd, red) to 227°for HIL, molecule 2 (8fab, blue). Also shown are Fabs NC6.8, unliganded (190 °, 1cgs, cyan) and liganded (153", 2cgr, yellow) and DB3, (182", 1dba, green). N M R has also been used to assess the dynamic properties o f an Fv or a single chain Fv of the phosphoryl-choline binding antibody McPC603 [65]. The N M R study o f the McPC603 single chain Fv indicates that there is some flexibility corresponding to rapid motion in the protein backbone that is localized to certain positions in the CDRs as well as to the linker and carboxy-terminus o f the Fv constant domain.
In a comparison between the N M R and crystal structure o f a trisaccharide-antibody complex [66], the authors have concluded that the antibody imposes an induced fit on the carbohydrate antigen, as the bound carbohydrate structure itself is not highly populated in solution. An N M R study of the isolated VL domain of 26-10 [67] shows only small differences compared with with the Xray structure o f the intact Fab domain [49].
Role of water in antibody-antigen recognition The most exhaustive analysis o f bound waters in and around an antibody combining site is that of the 1.8A resolution structures of the unliganded and liganded forms o f the anti-lysozyme Fv D1.3 [50"]. Rather surprisingly, a greater number, 45, of well ordered waters are located in the lysozyme-bound Fv than in its unliganded form, 23. Consistent with there being no net loss o f water molecules, titration calorimetry results suggest enthalpy drives the antibody-antigen interaction [50°]. Thus, these high-resolution studies have shown a clear role for water molecules in mediating antibody-antigen interaction, contrary to the earlier lower-resolution studies of antibody-antigen complexes. In a mutant o f D1.3, where VL Trp92 is replaced by aspartate [68], two water molecules occupy the space where the indole ring previously bound. This mutation, however, resulted in a decrease in the binding constant of three orders of mag-
nitude (108 to 105M-1). A similar addition of a water molecule was noted in antibody 26/9 [39], which is closely related to the previously determined 17/9 antibody [12,15°]; in 17/9, a change in the binding site at V L Asn94 (in 17/9) to histidine (in 26/9) resulted in a slight alteration of the bound ligand conformation, but no loss in affinity. Seven ordered water molecules have also been seen to play a role in the 2.2 A NC6.8 complex; they form bridges between the hgand and Fab [16]. Another study o f an anti-lysozyme antibody, H y H E L 5, by isothermal titration calorimetry, also showed an enthalpy-driven reaction [69].
Cross-reactivity Several recent studies have addressed the issue of how antibodies can cross-react with different antigens and vice versa. An excellent example of how two different antibodies bind to the same antigenic determinant is provided by the structures of N C l 0 and NC41 bound to influenza virus neuraminidase [53"]. These two antibodies bind quite differently to neuraminidase (Fig. 3) and provide a strong element of warning about prediction of antibody interactions from sequence comparisons. NC10 and NC41 share the same C D R H1 sequence, yet there is no correspondence in their interaction with the neuraminidase. Indeed, in NC10, the C D R H1 does not even contact neuraminidase [53"]. However, there is some conservation of the types o f amino acids that contact the neuraminidase, even though they are not related in sequence in each antibody. A comparison o f two closely related anti-influenza hemagglutinin peptide antibodies, 17/9 and 26/9, showed that minor changes in sequence can be accommodated in the antibody combining site without alteration in affinity for substrate or substantial difference in the bound peptide ligand conformation [39]. A detailed analysis o f peptides of different lengths bound to the 17/9 antibody showed that they bound in essentially the same conformation [15"].
Antibody-antigen interactions Wilson and Stanfield
(a)
(b)
Fig. 3. Stereoviews of Fabs NC41 and
t
Another interesting description of antibody cross-reactivity comes from the anti-progesterone antibody DB3, which binds differently shaped steroid antigens with nanomolar affinities [51°']. This study suggests that the binding site has good, but limited, complementarity to these high-affinity ligands and can accommodate different orientations of the steroid antigen. The structure of Fab D 11.15 [70], raised against hen egg white lysozyme (HEL), has been solved in complex with pheasant egg lysozyme (PHL), for which it actually has a higher affinity. This Fab seems to be able to recognize a broad spectrum of lysozymes, as it binds to a fairly conserved region on lysozyme, where most sequence changes occur at the edges of the epitope and do not adversely affect
NCl0, both bound to neraminidase. (a; top) Stereoview of Fab NC41 with bound neuraminidase. The neuraminidase is on top in very dark lines, with the Fab underneath, oriented so that the light chain is on the left. The residues that contact Fabs NC41 and NC10 are highlighted by shaded spheres (Pro328 and Asn329, darkest; Asn344, Ile366, Ile368, Ala369, and Ser370, medium; Asn400, Thr401, and Trp403, lightest). (b;bottom) Stereoview of Fab NCl0 with bound neuraminidase. The constant region of the Fab is disordered in this crystal structure. Gray scaling is the same as in (a). The neuraminidase epitopes for NC10 and NC41 overlap, but the two antibodies bind the same e.pitope in different ways.
binding. The Fab CHA255 was also solved in complex with two different metal chelate haptens [54], differing in affinity approximately 20-fold. A new idiotope-anti-idiotope Fab complex has provided a fascinating insight into the mimicry of antigens by antiidiotypic antibodies (Ab2) [71°]. Although the tertiary structure of the E2 glycoprotein antigen of feline infectious peritonitis virus is unknown, two sequences of six residues in antibody C D K s L1 and H1 correspond almost exactly to the antigen sequence of the E2 peplomer. These two anti-idiotypic antibody C D R s have major contacts to the antibody and may indeed be mimics of the original antigen. An earlier study on an
863
864
Proteins Table 3. Hyperviable loops used in antigen binding showing the percentage of the buried surface between antigen and antibody Fab DB3 DB3 DB3 DB3 DB3 CHA255 CHA255 AN02 1 F7 26-10 26-10 4-4-20 NC6.8 50.1 50.1 1 7/9 1 7/9 1 7/9 17/9 17/9 26/9 TE33 B1312 BV04-01 Se155-4 Se155-4 Se155-4 Se155-4 NC41 NC41 NC41 NC41 D1.3 D1.3-Fv D l l .15 HyHEL5 HyH EL5 HyHEL10 Je142 Average
PDB code 1dbb ldbj 1dbk 1d b m 2dbl 1 ind 1 ine 1baf 1fig 1 igj 1gj 4fab 2cgr 1ggi 1ggi 1him 1him 1 hin 1 ifh 1 ifh 1frg 1tet 2igf 1cbv 1mfb 1mfc 1mfd 1mfe 1nca 1ncb 1ncc 1ncd 1fdl 1vfb ljhl 2hfl 2iff 3hfm ljel
L1 (%)
L2 (%)
L3 (%)
4.0 0.0 0.0 2.8 7.7 12.7 11.2 14.3 0.0 0.4 0.7 30.4 12.5 9.3 7.4 5.0 5.0 6.5 5.3 5.3 5.5 28.7 8.1 21.1 23.3 6.2 10.0 10.2 1.0 1.6 3.2 1.4 9.0 12.4 7.4 12.7 13.1 11.8 26.2 9.3
0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.3 0.0 0.0 0.0 0.0 0.0 8.4 5,2 0.0 0.0 0.0 0.0 0.0 0.0 2.4 0.0 0.7 0.0 0.0 0.0 0.0 22.0 23.3 23.2 23.3 13.8 11.9 2.7 1,7 1.9 11.8 0.0 4.0
26.9 26.0 25.9 24.3 33.5 28.1 27.0 37.6 10.1 21.1 22.1 12.0 21.4 17.5 19.3 21.0 24.9 18.6 19.9 19.9 25.9 11.2 13.7 20.5 25.1 23.7 27.5 29.3 16.5 16.4 14.7 16.8 17.0 1 7.4 22.9 25.9 26.1 16.9 5.8 21.4
H1 (%)
H2 (%)
5.6 8.1 7.2 7.2 6.1 8.0 9.9 0.4 23.7 16.2 15.9 19.9 15.4 1.3 0.9 0.5 0.2 1.3 1.1 1.1 1.6 12.7 9.9 5.4 16.5 20.6 22.7 19.8 11.4 11.2 11.1 11.3 10.0 10.8 1 7.7 13.7 14.6 18.2 11.6 10.1
16.9 20.0 22.1 23.0 13.0 30.9 31.2 4.7 3.8 21.4 23.1 5.8 15.1 27.4 28.7 43.2 38.4 39.5 38.9 38.9 37.7 16.8 34.2 19.0 20.1 29.1 17.4 15.2 13.0 11.5 11.4 13.0 19.2 16.1 22.3 26.8 23.9 25.2 27.0 22.6
Framework (%) 40.7 39.0 38.3 38.9 34.4 20.3 20.5 32.0 61.2 35.3 33.1 31.8 34.3 22.5 23.6 30.3 31.5 33.7 34.8 34.8 29.2 27.1 32.2 33.4 15.0 20.3 22.3 23.3 28.4 29.1 28.5 27.1 26.4 26.5 24.0 15.4 14.3 8.6 27.7 29.0
5.9 6.9 6.6 3.9 5.3 0.0 0.1 4.7 1.3 5.6 5.2 0.0 1,4 13.6 14.9 0.0 0.0 0.4 0.0 0.0 0.0 1.1 1.9 0.0 0.0 0.0 0.0 2.1 7.7 6.9 7.9 7.0 4.7 4.9 3.0 3.9 6.0 7.4 1.6 3.5
aThe percentage of the buried surface between antigen and antibody resulting from each loop was calculated with the program MS [84] with a probe radius of 1.7A. CDRs are defined as in [28]. PDB code indicates protein databank entry.
angiotensin II-Ab3 complex showed that the bound angiotensin structure resembled a canonical L3 structure and suggested that the Ab2 antibody that was used to produce the Ab3 [30] mimiced angiotensin mainly through a single hypervariable loop. These results differ from the lysozyme Fab D1.3-Fab E225 idiotope-antiidiotope structure [72], where there was no obvious mimicry of the lysozyme antigen by the anti-idiotypic antibody [73,74].
Conclusions
The prediction of antibody structures has suffered a major setback this past year due to the large conformational changes that have now been seen in antibody structures on ligand binding [8°,17°°,52]. A more reassuring trend has been the agreement between the predicted conformation of individual CDRs with their actual X-ray structures, with the exception of C D R H3. To date,
Antibody-antigen interactionsWilson and Stanfield 865 no actual changes in canonical loop conformation have been seen on going from the unliganded to the liganded forms, with the exception o f CDP,. H3, which is very diverse in conformation as well as length and sequence. Indeed, it would appear that C D R H3 still ranks as the most important loop for binding and conformational variability; in contrast L2 is often not used in binding to smaller antigens [17••,31], even when all the other loops are used for binding (Table 3). The interaction of the three hypervariable loops, L3 (21%), H2 (23%) and H3 (29%) dominate the overall binding interactions compared with L1 (9%), L2 (4%) and H1 (10%) and the framework (4%). Two structures of humanized anti-CD18 antibodies (Fv and Fab fragments) have implicated a new region o f antigen contact at the carboxy-terminal part of H2 (residues 59-68) that has not been observed previously [75]. Finally, on the antibody engineering front, the structure o f a recombinant anti-sialidase (neuraminidase) single chain Fv [76] has shown interactions with neuraminidase similar to those seen for the NC10 Fab, but with no discernible electron density for the (Gly4Ser)3 single chain Fv linker. The brevity o f this article has made it impossible to provide an in-depth review of all the antibody structures determined in the past year. For other useful recent reviews, consuk [41,47,52,77-80].
Acknowledgements We thank Drs Robyn Malby and Peter Colman for NC10 coordinates and Wayne Zhou and Robert Fletterick for 17E8 coordinates. Research in the authors' laboratory is supported by NIH grants GM38419, GM46192 and GM49497 (to IAW). RLS is a Scholar of the American Foundation for Aids R e search. This is manuscript number 8946-MB from The Scripps Research Institute.
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IA Wilson and ILL Stanfield, MB13, Department of Molecular Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.
867
During the past year, 23 reports o f new antibody or antibody binding fragment (Fab) structures have appeared (Table 1), bringing the current total o f such structures to well over 50; about 40 of these are currently deposited in the Brookhaven Protein Data Base [1]. The growing number o f structures has led to more statistically valid conclusions about the nature o f antibody-antigen recognition, while continuing to provide some new surprises about features o f this interaction. We now have a clearer understanding o f the atomic details o f antigen binding to antibody, as well as of the role and extent o f conformational changes in this molecular recognition event. The accumulation o f structures is also useful for the prediction and determination of new antibody structures, for use in chemistry, biology, and medicine. Perhaps the most interesting new results that emerged during the review period were the first glimpses o f the three-dimensional architecture of three catalytic antibodies. Whereas natural enzymes have had a very long time to evolve their catalytic function, antibodies that catalyze chemical reactions (abzymes) can be produced in the laboratory in only a few weeks. While the first abzymes catalyzed only simple hydrolytic reactions, the present generation can carry out transformations that are disfavored or for which no natural enzyme catalyst is known [2-5]. Two o f the recently determined abzyme structures illustrate the different mechanisms they use in their catalysis [6",7"°]. Other major advances reported during this period concern the nature and role of conformational changes in antibody-antigen recognition. It is quite clear now that there is a large variation in the extent to which conformational changes occur in individual antibodies. Undoubtedly, further examples that extend the range and size o f these changes will be discovered [8°]. Still at issue is whether one can describe a universal mechanism for antibody-antigen recognition. The
Fab
CNJ206
Antigen
p-nitrophenyl
Unliganded
Liganded
Resolution
Reference
+
-
3.0
[29]
ester 17E8
Norleucine
-
+
2.5
[7 ° ° ]
1F7
phosphonate Transition state
-
+
3.0
[6 e°]
58.2
analogue gp120
-
+
3.0
[32 eel
peptide 17/9
Hmg peptide
+
+
2.8
[15 e]
TE33
CTP3 peptide HRV2 peptide PHL FIPV antibody
-
+
2.3
[31]
+
+
2.5
[17 ° ° ]
-
+ +
2.4 2.9
[70] [71 e]
+
+
2.7
[1, 5, 13,
-
+
2.2, 2.8
[54]
8F5 Dl1.15 409.5.3 DB3 CHA255
Steroid haptens Metal chelate
Je172
haptens DNA
+
2.7
[81]
Je1318 NC6.8
DNA Sweetening
+ +
+
2.8 2.6, 2.2
[82] [16]
50.1
compound gp120
+
+
2.8
[8#,12]
peptide D1.3 Fv
HEL
+
+
1.8
[50e]
D1.3
HEL
-
+
1.8
[68]
mutant 26-10
Digoxin
-
+
2.7, 2.5
[49]
N C l 0 scFv NC10
Neuraminidase Neuraminidase
-
+ +
3.0 2.5
[76] [53 e°] [46]
17-1A
HRVl 4
+
-
2.7
HuH52-OZ
CDI 8
+
-
3.0
[75]
HuH52-AA Fv
CD18
+
-
1.9
[75]
Hmg, hemagglutinin; PHL, pheasant egg lysozyme; FIPV, feline infectious
peritonitus virus; HEL, hen egg lysozyme.
current trend favors induced fit [9-13], and some new examples [8°,14°',15°,16,17 °°] add support to that hy-
Abbreviations
Ab2--anti-idiotypic antibody; Fab---antibody binding fragment; Fv--Fab variable domain. © Current Biology Ltd ISSN 0959-440X
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Proteins pothesis. These examples still do not constitute proof, however, as the X-ray structures represent single snapshots of a dynamic situation. High-resolution N M R studies, combined with detailed kinetic and thermodynamic measurements and molecular dynamic simulations, are now providing welcome new information to help resolve these issues. In the end, it may turn out that antibody-antigen recognition cannot be explained by a simple model, such as for peptide antigen recognition by MHC class I [18-20] and M H C class II [21,22] molecules. Undoubtedly, the most controversial paper in this period [16] revisits the question of whether antigen binding causes a signal to be transmitted from the antibody combining site to the more distal constant domains, thereby initiating the effector response that includes complement and Fc receptor binding. For such a complicated problem, both the uncleaved, unliganded antibodies and their complexes probably need to be crystallized and studied. The recently determined X-ray structure of an intact IgG that contains a normal, rather than deleted, hinge region shows there is still some promise for this approach [23].
Catalytic antibodies Several years after the initial demonstration that chemical reactions can be catalyzed by antibodies produced
through immunization of mice with analogues of the transition state of the reaction [24-27], a flurry of activity has resulted in the structural determinations of several abzymes. The crystal structure of an antibody (1F7) that catalyzes a chorismate mutase transformation shows that its endo-oxabicyclic dicarboxylic acid transition state analogue (TSA) is bound in a small but highly complementary depression on the antibody surface [6"] (Fig. la). The antibody does not appear to require any functional groups to participate in the catalysis, and therefore seems to work mainly by restricting the substrate to a conformation that mimics its higher energy, pericychc transition state. A second abzyme crystal structure [7"'] (Fig. lb) supports the notion that catalytic antibodies may mimic natural enzymes by containing arrangements of catalytic groups that resemble those found in known enzyme structures. On the basis of the X-ray structure of 17E8 (an active hydrolytic antibody) in combination with kinetic data, Zhou et al. [7°°] propose that the mechanism of hydrolysis by the antibody is similar to that found in serine proteases. Indeed, the antibody combining site contains two residues from the heavy chain (His H35 and SetH95; numbering as in [28]) that appear to be topologically similar to the equivalent components of the Ser-His-Asp catalytic triad in serine proteases. AdditionaUy, the residue LysH93 from the antibody serves as the 'oxyanion hole'. In the crystal structure, the hydroxyl group of Set H95 is rather far (5-6 A) from the phospho-
(a)
(b)
Fig. 1. (a; top) Stereoview of the catalytic Fab 1F7. The binding site for 1F7 is shown with the bound transition state analogue. The light chain is on the left. (b; bottom) Catalytic Fab 17E8. The hydrolytic Fab with antigen is in the same orientation as 1F7 above. All figures created with MOLSCRIPT [83].
Antibody-antigen interactions Wilson and Stanfield rus atom of the hapten and makes a hydrogen bond with a neighboring TyrH97. Zhou et al. [7°°] show that rotations of both the serine and histidine sidechains can put them into position to carry out their proposed function. A structure determination of 17E8 at the pH optimum of the enzymatic reaction (around pH 9.0) is currently underway to test this hypothesis (TS Scanlan, personal communication). It is also interesting to look at the frequency of histidine and serine at these positions in the variable region of IgG heavy chains. Histidine is the residue found most frequently at position H35 (in 378/1459 protein sequences studied), whereas aspartate is the' residue found most frequently at position H95 (in 289/1451 cases); serine is found at position H95 less frequently, in 187 of 1451 sequences studied [28]. A third structure of an unliganded Fab with esterolytic activity (CNJ206) [29] reveals the binding site for the para-nitrophenyl ester substrate. On the basis of a structural model for the complex with the transition state analogue, the authors [29] propose that catalysis also involves an oxyanion hole similar to that found in serine protease active sites (and in Fab 17E8). Whether such details of ligand binding can be modeled from the unliganded structure obviously awaits the X-ray structure determination of the ligand-bound form of the antibody.
Antibody-peptide complexes The feature most commonly seen in antibody-peptide complex structures is a ~-turn in the peptide that is deeply embedded in the antibody-antigen interface. Several examples of this recognition motif now exist and inClude a type II [~-turn (VPHK, residues 71-74; singleletter amino acid code) in a peptide from myohemerythrin [10], a type I [~-turn (DYAS, residues 104-107) in an influenza hemagglutinin peptide [12], and two tight turns in the peptide hormone angiotensin II [30]. Three new examples from this review period are a type II ~turn in the cholera toxin peptide CTP3 bound to Fab TE33, (VPGS, residues 52-55; [31]), a type I ~-turn in the VP2 peptide from human rhinovirus 2 (HR.V2) bound to Fab 8F5 (AETP,.,2158-2161; [17°°]) and a type II ~-turn in a peptide from human immunodeficiency virus 1 (HIV-1) gpl20 bound to Fab 59.1 (GPGk, residues 319-322; [32°°]). These last two examples actually contain more complicated turn arrangements than those mentioned above. In the 8F5-VP2 complex, the peptide (VKAETRLNPDLQPTE-NH2, residues 2156-2170) contains an asparagine-pseudoturn (NPD) following the [~-turn. The HIV-1 peptide in the 59.1 complex has an unexpected double turn, where the predicted type II ~-turn [33] is followed by a combined type III (GR_AF)- type I (RAFY) turn. In all these structures, anywhere from 7 to 12 of the peptide residues are ordered, no matter how long the peptide in the crystal (up to 24 residues in the 59.1 complex). Thus, the antibody interactions must extend beyond the [3-turns, and include extended ~-strand type interactions between the peptide and antibody in
the 17/9-hemagglutinin [12,15°], TE33-cholera toxin [31] and 8F5-VP2 complexes [17°']. The only example that does not involve interaction with a tight turn is the Fab 50.1-HIV-1 peptide complex, in which the peptide is essentially extended and runs along a 25 ]x groove between the variable light and heavy domains (VL and VH) [34]. Even in this structure, however the carboxyterminal end of the peptide starts to adopt a turn-like structure before exiting the binding site. In the cholera toxin peptide paper [31], Shoham suggests that "antipeptide antibodies seem to induce a ~turn conformation in the bound peptide irrespective of the sequence of the peptide" and cites as examples the B1312 and 17/9 structures [10,12]. Secondary structure predictions for these peptide sequences, however, show that they have a marked tendency to form the turn types found in the Fab-peptide structures, as in the recent examples of the G P G R sequence (predicted to be type II) in the HIV-1 peptide and VPGS (predicted to be type II) in the cholera toxin CTP3 peptide. Therefore, it is not likely that the antibody randomly induces peptides to adopt ~-turns. O f more relevance is how the bound peptide structures correspond to their cognate structures in their respective proteins. The myohemerythrin (B1312) and cholera toxin (TE33) anti-peptide antibodies do not bind to the native versions of the proteins, except in solid-phase enzyme-linked immunosorbent assay (ELISA) systems (TE33; [31,35]). However, they may react with nonnative forms, such as the binding of apomyohemerythrin by B1312 [10]. The myohemerythrin peptide sequence in holomyohemerythrin is largely helical [36], whereas the peptide sequence in the intact cholera toxin B-subunit [37] has a type I ~-turn at residues Gly-SerGlu-His (positions 54-57) and an or-helix from residues 59-64. On the other hand, three anti-viral antibodies (50.1 and 59.1, against HIV-1 gp120), and 8F5, against (I-IRV2 VP2) neutralize viral infectivity and presumably react with native protein. The anti-influenza antibody 17/9 and several related antibodies bind to the biologically important fusion form of influenza virus hemagglutinin at low pH [38]; the rhinovirus antibody was raised against native virions [17 °°] and the HIV-1 gpl20 antibodies neutralize and inhibit various viral functions [32°°]. Hence, the influenza hemagglutinin, rhinovirus VP2, and gpl20 peptides would be expected to mimic their protein counterparts [12,15 °, 17°°,34]. An extensive study of various hemagglutinin peptides in complex with Fabs 17/9 [15 °] and 26/9 [39] shows that the only major difference between the antibody-bound peptide conformation and its counterpart in the intact hemagglutinin is in the ~ angle of residue Prol03. Two sequence changes in the binding site between the closely related 26/9 and 17/9 antibodies leave the overall bound peptide conformation virtually unchanged except for an additional interaction with a water molecule [39]. Although the structure for the 8F5 antigen, HRV2, has not been determined, the related HRV1 structure contains a loop that, despite its difference in sequence, is very similar to the corresponding HP,.V2 peptide structure, except for a
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Proteins cis-proline (KAETRLNPDLQ). The structure for HIV1 gp120 is not yet known, but an N M R structure o f the 40 amino acid loop containing the sequence of the peptides bound to Fabs 50.1 and 59.1 also indicates that the G P G R sequence tends to form a a-turn [40]. Thus, it is clear that antibodies generated against peptides or peptide analogues (17/9, 26/9, 50.1, 59.1), or the intact virion (8F5), can cross-react with both peptide and protein and hence can provide valuable information for vaccine development or drug design [32°°]. Indeed, the structures o f the two Fab complexes o f 50.1 and 59.1 with different but overlapping peptides o f the V3 loop of HIV-1 gp120 have enabled a conformation for the top of the V3 loop to be assembled by stitching together the structures of the two peptide pieces. A more comprehensive recent analysis of peptide-antibody complex structures can be found elsewhere [41].
Virus-antibody structures A combination of electron microscopy and X-ray diffraction is now being used to elucidate the details of how neutralizing antibodies bind to viruses. A pioneering study on a cowpea mosaic virus with an Fab fragment of a neutralizing antibody, using cryo-electron microscopy and image reconstruction at 23 fi~ [42], showed the footprint of the Fab on the viral capsid protein. A recent study [43] with the intact IgG showed monodentate binding to the cowpea mosaic virus, which contrasts with the bidentate binding to rhinovirus 14 (HRV14) by the antibody 17-IA [44",45]. The Fab and the whole IgG o f l 7 - I A both bind across the icosahedral twofold around the fivefold axes; an X-ray structure o f the Fab fragment of this antibody [46] has now enabled a better fit to be made for the image reconstruction. It suggests that the virus itself does not change its gross conformation on binding of Fab or IgG [44°,45]. More details of how intact viruses are recognized by monoclonal antibodies are likely to come soon from the combination of X-ray diffraction and electron microscopy.
Conformational changes Several types, of conformational rearrangements have been documented in Fab fragments in response to ligand binding. In our previous review in Current Opinion in Structural Biology [47], the changes outlined included small side-chain rearrangements, segmental movements of complementarity determining region loops (CDRs; L1, L2, L3 and H1, H2, H3 from the light and heavy chain respectively), large rearrangements o f C D R H3 and alterations in the relative disposition o f the VL-V H domains. The Fabs 17/9 [12] and BV04-01 [11] exhibited the largest of these changes in CDtk H3 (3-5 ,~ in backbone atoms for 17/9) and in relative VL-V H rotation (7.5 ° for BV04-01). An even larger relative rotation (16 °) of the VL-V H domains has now been observed for the anti-HIV-1 Fab 50.1 on complex formation with the V3 loop peptide [8°] which results in a significantly altered combining site in the peptide-bound Fab. Super-
imposed on this quaternary structural change is the now commonly seen rearrangement o f the H3 loop, which moves out o f the binding site with root mean square deviations o f up to 5 A in backbone atoms and 6A in side-chain atoms compared to the unliganded Fab. A detailed analysis o f the somewhat limited database containing both unliganded and liganded Fabs indicates that the larger domain rotations are associated, perhaps not surprisingly, with smaller buried surface areas (1000A2) between the VL and VH domains, compared to smaller domain rotations (0°-4 ° rotations) that have VL-V H buried surface areas of up to 1700 A2 [8"]. Thus, a smaller VL-V H interface may be more likely to be subject to such quaternary rearrangements on ligand association. These results constitute experimental proof of the interface adaptor hypothesis put forward by Colman [48], who proposed that when the size of the antigen interface approaches the size of the VL-V H interdomain interface, then VL-V H rearrangements might accompany and play a role in antibody-antigen complementarity. Other examples o f unliganded and liganded structures reported during this review period include the antisweetener Fab NC6.8 [16], the anti-rhinovirus Fab 8F5 [17"'], the anti-digoxin antibody 26-10 [49], the highresolution refinement o f the anti-lysozyme Fv (variable domain o f the Fab) D1.3 [50], and the steroid binding Fab DB3 [14"°,51°']. These Fabs and Fvs show varying VL-Vkl rotations upon ligand association: 2.8 ° (NC6.8, 1295A2 VL--VH interface size); 0 ° (DB3, 1425fi~2); 2 ° (26-10, 1637fi~2); and 2.2 ° (D1.3 Fv, 1469fi~2). Thus at present, the majority OfVL-V H rearrangements are clustered around 2-6 °, with only BV04-01 and 50.1 showing larger rotations. A review of the impact of these changes on structure prediction and design of antibodies has been discussed elsewhere [52]. Fabs are also very flexible at the junction between the variable (V) and the constant (C) superdomains; in one recent Fab structure (NC10), only the Fv (VL-VH) is ordered in the crystal [53"']. The so-called elbow angle is a measure o f the relative disposition of these superdomains and corresponds to the angle between the two pseudo twofold rotation axes of the V and C superdomains (Fig. 2). The range o f elbow angles increased during the review period and now varies from 127 ° for unliganded Fab 8F5 [17"] to 194 ° for Fab CHA255 [54] (Table 2), although a set o f unpublished coordinates which have been deposited in the Brookhaven Protein Data Bank for Fab HIL (pdb8fab.ent; FA Saul, tkJ Poljak, depositors) has an elbow angle of 227 ° for one of the two unliganded molecules in the HIL structure. There are now several examples of two Fabs in the same crystallographic asymmetric unit having different elbow angles, both for unliganded Fabs (2130 and 227 ° for HIL [pdb8fab.ent] and 159 ° and 167 ° for Je142 [55]) and for liganded Fabs (164 ° and 177 ° for 50.1 [34]). Differences in elbow angles have also been reported between unliganded and liganded structures of the same Fab (161 ° and 176 ° for 17/9 [12], 176 ° and 164 ° for 50.1 [8°], and 190 ° and 153 ° for NC6.8 [16]). The last example, the anti-sweetener Fab, NC6.8, prompted Guddat et
Antibody-antigen interactions Wilson and Stanfield al. [16] to revisit an old theory about signal transmission
from the variable to the constant domain upon hgand binding. This idea was popular in the mid-1970s [56], when a closed or bent elbow angle was thought to be characteristic of antigen-bound antibodies and an open or straight elbow angle (approaching 180 °) characteristic of unliganded antibodies. Not until the solution of the first Fab-protein complex structure (D1.3-1ysozyme [57]) was this suggestion disproved [58]; however, Huber did not completely rule out the idea of "elbow bending as a signal transfer mechanism" [58], which had also been suggested from light chain dimer studies [59]. Table 2. Elbow angles. Unliganded Fab
PDBa code
Angle (0) Mol 1 Mol 2
Liganded Fab
PDB code
Angle (°) Mol 1 Mol 2
8F5* NEW* McPC603* B72.3" J539" HC19" YST9.1* hu4DS-ver4* hu4DS-ver7* KOL* 36-71" 3D6" R19.9" HIL*
lbbd 7fab 2mcp lbbj 2fbj 1gig 1roam lfvd 1fve 2fb4 6lab ldtb lfai 8lab
127.3 130.2 133.3 137.6 143.9 146,7 150.9 156.9 155,3 1 5 7 . 1 154.8 166.9 167.0 176.1 t76,8 212.9 226.9
1F7 59.1 TE33 HyHEL10 NC41 NC4i NC41 NC41 AN02 HyHEL-5 HyHEL-5 D1.3 26/9 Se155-4 Se155-4 Se155-4 Set55-4 4-4-20 CHA255 CHA255
1fig lacy ltet 3hfm lnca tncb lncc lncd 1baf 2iff 2hfl lfdl lfrg lmfe 1mfb 1mfd lmfc 4lab lind line
132.3 134.8 141.2 146.4 147.1 t47.4 147.9 147.9 154.2 16t.9 162.5 173.8 175.3 183.9 184.0 184.0 184.8 186.0 194.2 194.3
B1312 Je142 17/9 17/9 17/9 50.1 50.1 DB3 D83 DB3 DB3 DB3 26-10 BV04-01 NC6.8
2igf I jel lhim thin lifh Iggi Iggi Idbb Idbj Idbk ldbm 2dbt ] igj lcbv 2cgr
157.4 154.9 174,2 177.3 175,9 163.9 163,9 182.6 182.5 182.6 182.5 182.3 183.9 175.5 152.9
81312 Je142 17/9 17/9 17/9 50.I 50.1 DB3 DB3 DB3 DB3 DB3 26-10 BV04-01 NC6.8
ligf b lhil lhil lhil Iggb Iggc Idba Idba Idba ldba tdba 1igi lnbv 1cgs
154.3 159 161.2 161.2 161.2 173.5 175.6 181.7 181.7 181.7 181.7 181.7 186.2 188.2 189.8
156.6 167 161.8 161,8 161.8
173.1
176.5 176.5
184.0
apdb code indicates the protein databank entry information. Unliganded Fab elbow angles for which corresponding liganded Fab elbow angles have not been determined are asterisked (*). bNot deposited in the protein data bank,
Currently, such internal variations in elbow angles have been documented only with Fab structures, although a cryo-electron microscopy analysis of the neutralizing antibody 17-IA bound to human rhinovirus 14 suggests that the bidentate IgG has an elbow angle different from the Fab fragment alone [44°]. Thus, it is somewhat surprising that the NC6.8 structure mentioned above has been used to invoke the transmission theory, given that only one of the ligand complexes, a trisubstituted guani-
dine hapten bound to NC6.8, crystallizes with a different elbow angle, whereas two others appear to resemble the unliganded Fab NC6.8. By the authors own calculations, other structures, such as Fab 17/9, do not conform to their more detailed concerted shifts ideas. Clearly, more studies with intact immunoglobulins are needed to resolve such questions of transmitted conformational changes. Indeed, the ligand-induced alterations in VL-V H domain association would seem to be strong candidates for such a signal, but, wisely, no groups have (yet!) come to that conclusion. Further confirmation of flexibility elsewhere in immunoglobnlin molecules was provided by a recent [13C]NMR. study of a murine intact monoclonal IgG2a [60] and by the cryo-electron microscopy study of intact IgGs bound to human rhinovirus 14 [44",45]. In the N M R study, the hinge region connecting CH1 to CH2 was described as a mosaic structure of three components: the upper hinge, a structure including the linked disulfides, and the lower hinge. In the rhinovirus-IgG complex structure, only the Fab fragments could be seen in the image reconstruction for the 30 IgGs that straddle the icosahedral twofold axis of the virus and bind to the VP1 subunits that surround the fivefold axes. These studies confirm the flexibility of the hinge region that was observed early on in the intact Kol structure, in which the Fc region was disordered [56]. Since then, the only intact antibody structures to have been determined are those with deleted hinge regions, such as Dob [61] and Mcg [62], and for an IgG with a normal hinge that surprisingly contained an ordered Fc region [23]. Questions about the number of conformational states that an antibody can adopt were answered in part with the structure determination of five complexes of anti-progesterone Fab' DB3 [14°',51 ''] with different steroids. Two classes of steroid ligands that differ substantially in their shapes were found to bind in alternative orientations in the antibody combining site. The Fab' made minor adjustments in its conformation (0.35 A root mean square deviation for backbone atoms among the steroid-bound forms) in order to accommodate these differently shaped ligands. N M R studies X-ray structures have suggested the existence of distinct unhganded and hganded forms of Fabs [47,52], with major conformational variation occuring in the C D R H3 loop. There are no well documented examples of the other C D R loops adopting substantially different structures or varying from the canonical forms [63] when going from their unliganded to liganded forms. Some X-ray evidence suggests that the C D R L1 can be more flexible at its top [64]; otherwise, there are no obvious trends in decreased mobility in the CDl
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Proteins
Fig. 2. Elbow angles. In this stere0view, several Fabs have been superimposed by their variable domains, so the differences in their elbow angles are visible. The current range of elbow angles ranges from 127°for 8F5 (1 bbd, red) to 227°for HIL, molecule 2 (8fab, blue). Also shown are Fabs NC6.8, unliganded (190 °, 1cgs, cyan) and liganded (153", 2cgr, yellow) and DB3, (182", 1dba, green). N M R has also been used to assess the dynamic properties o f an Fv or a single chain Fv of the phosphoryl-choline binding antibody McPC603 [65]. The N M R study o f the McPC603 single chain Fv indicates that there is some flexibility corresponding to rapid motion in the protein backbone that is localized to certain positions in the CDRs as well as to the linker and carboxy-terminus o f the Fv constant domain.
In a comparison between the N M R and crystal structure o f a trisaccharide-antibody complex [66], the authors have concluded that the antibody imposes an induced fit on the carbohydrate antigen, as the bound carbohydrate structure itself is not highly populated in solution. An N M R study of the isolated VL domain of 26-10 [67] shows only small differences compared with with the Xray structure o f the intact Fab domain [49].
Role of water in antibody-antigen recognition The most exhaustive analysis o f bound waters in and around an antibody combining site is that of the 1.8A resolution structures of the unliganded and liganded forms o f the anti-lysozyme Fv D1.3 [50"]. Rather surprisingly, a greater number, 45, of well ordered waters are located in the lysozyme-bound Fv than in its unliganded form, 23. Consistent with there being no net loss o f water molecules, titration calorimetry results suggest enthalpy drives the antibody-antigen interaction [50°]. Thus, these high-resolution studies have shown a clear role for water molecules in mediating antibody-antigen interaction, contrary to the earlier lower-resolution studies of antibody-antigen complexes. In a mutant o f D1.3, where VL Trp92 is replaced by aspartate [68], two water molecules occupy the space where the indole ring previously bound. This mutation, however, resulted in a decrease in the binding constant of three orders of mag-
nitude (108 to 105M-1). A similar addition of a water molecule was noted in antibody 26/9 [39], which is closely related to the previously determined 17/9 antibody [12,15°]; in 17/9, a change in the binding site at V L Asn94 (in 17/9) to histidine (in 26/9) resulted in a slight alteration of the bound ligand conformation, but no loss in affinity. Seven ordered water molecules have also been seen to play a role in the 2.2 A NC6.8 complex; they form bridges between the hgand and Fab [16]. Another study o f an anti-lysozyme antibody, H y H E L 5, by isothermal titration calorimetry, also showed an enthalpy-driven reaction [69].
Cross-reactivity Several recent studies have addressed the issue of how antibodies can cross-react with different antigens and vice versa. An excellent example of how two different antibodies bind to the same antigenic determinant is provided by the structures of N C l 0 and NC41 bound to influenza virus neuraminidase [53"]. These two antibodies bind quite differently to neuraminidase (Fig. 3) and provide a strong element of warning about prediction of antibody interactions from sequence comparisons. NC10 and NC41 share the same C D R H1 sequence, yet there is no correspondence in their interaction with the neuraminidase. Indeed, in NC10, the C D R H1 does not even contact neuraminidase [53"]. However, there is some conservation of the types o f amino acids that contact the neuraminidase, even though they are not related in sequence in each antibody. A comparison o f two closely related anti-influenza hemagglutinin peptide antibodies, 17/9 and 26/9, showed that minor changes in sequence can be accommodated in the antibody combining site without alteration in affinity for substrate or substantial difference in the bound peptide ligand conformation [39]. A detailed analysis o f peptides of different lengths bound to the 17/9 antibody showed that they bound in essentially the same conformation [15"].
Antibody-antigen interactions Wilson and Stanfield
(a)
(b)
Fig. 3. Stereoviews of Fabs NC41 and
t
Another interesting description of antibody cross-reactivity comes from the anti-progesterone antibody DB3, which binds differently shaped steroid antigens with nanomolar affinities [51°']. This study suggests that the binding site has good, but limited, complementarity to these high-affinity ligands and can accommodate different orientations of the steroid antigen. The structure of Fab D 11.15 [70], raised against hen egg white lysozyme (HEL), has been solved in complex with pheasant egg lysozyme (PHL), for which it actually has a higher affinity. This Fab seems to be able to recognize a broad spectrum of lysozymes, as it binds to a fairly conserved region on lysozyme, where most sequence changes occur at the edges of the epitope and do not adversely affect
NCl0, both bound to neraminidase. (a; top) Stereoview of Fab NC41 with bound neuraminidase. The neuraminidase is on top in very dark lines, with the Fab underneath, oriented so that the light chain is on the left. The residues that contact Fabs NC41 and NC10 are highlighted by shaded spheres (Pro328 and Asn329, darkest; Asn344, Ile366, Ile368, Ala369, and Ser370, medium; Asn400, Thr401, and Trp403, lightest). (b;bottom) Stereoview of Fab NCl0 with bound neuraminidase. The constant region of the Fab is disordered in this crystal structure. Gray scaling is the same as in (a). The neuraminidase epitopes for NC10 and NC41 overlap, but the two antibodies bind the same e.pitope in different ways.
binding. The Fab CHA255 was also solved in complex with two different metal chelate haptens [54], differing in affinity approximately 20-fold. A new idiotope-anti-idiotope Fab complex has provided a fascinating insight into the mimicry of antigens by antiidiotypic antibodies (Ab2) [71°]. Although the tertiary structure of the E2 glycoprotein antigen of feline infectious peritonitis virus is unknown, two sequences of six residues in antibody C D K s L1 and H1 correspond almost exactly to the antigen sequence of the E2 peplomer. These two anti-idiotypic antibody C D R s have major contacts to the antibody and may indeed be mimics of the original antigen. An earlier study on an
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Proteins Table 3. Hyperviable loops used in antigen binding showing the percentage of the buried surface between antigen and antibody Fab DB3 DB3 DB3 DB3 DB3 CHA255 CHA255 AN02 1 F7 26-10 26-10 4-4-20 NC6.8 50.1 50.1 1 7/9 1 7/9 1 7/9 17/9 17/9 26/9 TE33 B1312 BV04-01 Se155-4 Se155-4 Se155-4 Se155-4 NC41 NC41 NC41 NC41 D1.3 D1.3-Fv D l l .15 HyHEL5 HyH EL5 HyHEL10 Je142 Average
PDB code 1dbb ldbj 1dbk 1d b m 2dbl 1 ind 1 ine 1baf 1fig 1 igj 1gj 4fab 2cgr 1ggi 1ggi 1him 1him 1 hin 1 ifh 1 ifh 1frg 1tet 2igf 1cbv 1mfb 1mfc 1mfd 1mfe 1nca 1ncb 1ncc 1ncd 1fdl 1vfb ljhl 2hfl 2iff 3hfm ljel
L1 (%)
L2 (%)
L3 (%)
4.0 0.0 0.0 2.8 7.7 12.7 11.2 14.3 0.0 0.4 0.7 30.4 12.5 9.3 7.4 5.0 5.0 6.5 5.3 5.3 5.5 28.7 8.1 21.1 23.3 6.2 10.0 10.2 1.0 1.6 3.2 1.4 9.0 12.4 7.4 12.7 13.1 11.8 26.2 9.3
0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.3 0.0 0.0 0.0 0.0 0.0 8.4 5,2 0.0 0.0 0.0 0.0 0.0 0.0 2.4 0.0 0.7 0.0 0.0 0.0 0.0 22.0 23.3 23.2 23.3 13.8 11.9 2.7 1,7 1.9 11.8 0.0 4.0
26.9 26.0 25.9 24.3 33.5 28.1 27.0 37.6 10.1 21.1 22.1 12.0 21.4 17.5 19.3 21.0 24.9 18.6 19.9 19.9 25.9 11.2 13.7 20.5 25.1 23.7 27.5 29.3 16.5 16.4 14.7 16.8 17.0 1 7.4 22.9 25.9 26.1 16.9 5.8 21.4
H1 (%)
H2 (%)
5.6 8.1 7.2 7.2 6.1 8.0 9.9 0.4 23.7 16.2 15.9 19.9 15.4 1.3 0.9 0.5 0.2 1.3 1.1 1.1 1.6 12.7 9.9 5.4 16.5 20.6 22.7 19.8 11.4 11.2 11.1 11.3 10.0 10.8 1 7.7 13.7 14.6 18.2 11.6 10.1
16.9 20.0 22.1 23.0 13.0 30.9 31.2 4.7 3.8 21.4 23.1 5.8 15.1 27.4 28.7 43.2 38.4 39.5 38.9 38.9 37.7 16.8 34.2 19.0 20.1 29.1 17.4 15.2 13.0 11.5 11.4 13.0 19.2 16.1 22.3 26.8 23.9 25.2 27.0 22.6
Framework (%) 40.7 39.0 38.3 38.9 34.4 20.3 20.5 32.0 61.2 35.3 33.1 31.8 34.3 22.5 23.6 30.3 31.5 33.7 34.8 34.8 29.2 27.1 32.2 33.4 15.0 20.3 22.3 23.3 28.4 29.1 28.5 27.1 26.4 26.5 24.0 15.4 14.3 8.6 27.7 29.0
5.9 6.9 6.6 3.9 5.3 0.0 0.1 4.7 1.3 5.6 5.2 0.0 1,4 13.6 14.9 0.0 0.0 0.4 0.0 0.0 0.0 1.1 1.9 0.0 0.0 0.0 0.0 2.1 7.7 6.9 7.9 7.0 4.7 4.9 3.0 3.9 6.0 7.4 1.6 3.5
aThe percentage of the buried surface between antigen and antibody resulting from each loop was calculated with the program MS [84] with a probe radius of 1.7A. CDRs are defined as in [28]. PDB code indicates protein databank entry.
angiotensin II-Ab3 complex showed that the bound angiotensin structure resembled a canonical L3 structure and suggested that the Ab2 antibody that was used to produce the Ab3 [30] mimiced angiotensin mainly through a single hypervariable loop. These results differ from the lysozyme Fab D1.3-Fab E225 idiotope-antiidiotope structure [72], where there was no obvious mimicry of the lysozyme antigen by the anti-idiotypic antibody [73,74].
Conclusions
The prediction of antibody structures has suffered a major setback this past year due to the large conformational changes that have now been seen in antibody structures on ligand binding [8°,17°°,52]. A more reassuring trend has been the agreement between the predicted conformation of individual CDRs with their actual X-ray structures, with the exception of C D R H3. To date,
Antibody-antigen interactionsWilson and Stanfield 865 no actual changes in canonical loop conformation have been seen on going from the unliganded to the liganded forms, with the exception o f CDP,. H3, which is very diverse in conformation as well as length and sequence. Indeed, it would appear that C D R H3 still ranks as the most important loop for binding and conformational variability; in contrast L2 is often not used in binding to smaller antigens [17••,31], even when all the other loops are used for binding (Table 3). The interaction of the three hypervariable loops, L3 (21%), H2 (23%) and H3 (29%) dominate the overall binding interactions compared with L1 (9%), L2 (4%) and H1 (10%) and the framework (4%). Two structures of humanized anti-CD18 antibodies (Fv and Fab fragments) have implicated a new region o f antigen contact at the carboxy-terminal part of H2 (residues 59-68) that has not been observed previously [75]. Finally, on the antibody engineering front, the structure o f a recombinant anti-sialidase (neuraminidase) single chain Fv [76] has shown interactions with neuraminidase similar to those seen for the NC10 Fab, but with no discernible electron density for the (Gly4Ser)3 single chain Fv linker. The brevity o f this article has made it impossible to provide an in-depth review of all the antibody structures determined in the past year. For other useful recent reviews, consuk [41,47,52,77-80].
Acknowledgements We thank Drs Robyn Malby and Peter Colman for NC10 coordinates and Wayne Zhou and Robert Fletterick for 17E8 coordinates. Research in the authors' laboratory is supported by NIH grants GM38419, GM46192 and GM49497 (to IAW). RLS is a Scholar of the American Foundation for Aids R e search. This is manuscript number 8946-MB from The Scripps Research Institute.
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IA Wilson and ILL Stanfield, MB13, Department of Molecular Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.
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