Diversity in Hapten Recognition: Structural Study of an Anti-cocaine Antibody M82G2

doi:10.1016/j.jmb.2005.03.080 J. Mol. Biol. (2005) 349, 570–582

Diversity in Hapten Recognition: Structural Study of anAnti-cocaine Antibody M82G2

Edwin Pozharski1, Aaron Moulin1, Anura Hewagama2

Armen B. Shanafelt2, Gregory A. Petsko1 and Dagmar Ringe1*

1Rosenstiel Basic MedicalSciences Research CenterBrandeis University, 415 SouthStreet, Waltham, MA 02454USA

2Roche Diagnostics Corporation9115 Hague Road, IndianapolisIN 46250, USA

0022-2836/$ - see front matter q 2005 P

Present addresses: A. HewagamaMichigan School of Medicine, Ann AA. B. Shanafelt, Eli Lilly and CompCenter, Indianapolis, IN 46285, USAAbbreviation used: CDR, comple

determining region.E-mail address of the correspond

[email protected]

Antibodies against cocaine and other drugs of abuse are the basis fordiagnostic tests for the presence of those drugs in human serum. The 1.7 Aresolution crystal structure of the anti-cocaine monoclonal antibodyM82G2 in complex with cocaine is presented. This structure determinationwas undertaken to establish the stereochemical features in the antibodybinding site that confer specificity for cocaine, and as part of an ongoingproject to understand the rules that govern molecular recognition. Thecocaine-binding site can be characterized topologically as a narrow grooveon the protein surface. The antibody utilizes water-mediated hydrogenbonding, and cation–p and stacking (p–p) interactions to providespecificity. Comparison with the previously published structure of theanti-cocaine antibody GNC92H2 shows that binding of a small ligandcan be achieved in diverse ways, both in terms of a binding sitestructure/topology and protein–ligand interactions.

q 2005 Published by Elsevier Ltd.

Keywords: protein–ligand interaction; cocaine; antibody engineering;immunotherapy; antibody structure
*Corresponding author
Introduction

The ability of the immune system to generatespecific antibodies for virtually any antigen hasbeen exploited in many different applications.Specifically, antibodies against small molecules(haptens) are used as catalysts,1 diagnostic testsystems2 and in immunopharmacotherapy.3 Inparticular, monoclonal antibodies have beendeveloped for the purposes of catalytic drugdegradation4 and active immunization5 in thetreatment of cocaine abuse. Antibodies for theseapplications are produced using hybridoma tech-nology, but their qualities could be furtherimproved through rational design if the structuraldeterminants of binding specificity were under-stood. The almost unlimited diversity of possibleantigens and the structural flexibility of antigen-binding sites make antibodies an outstanding

ublished by Elsevier Ltd.

, University ofrbor, MI 48109, USA;

any, Lilly Corporate.mentarity-

ing author:

model to study protein–ligand interactions ingeneral.

Thus, a better understanding of how antibodiesbind small ligands and what determines bindingaffinity and specificity would also greatly benefitthe broader field of structural studies of protein–ligand interactions and structure-based drugdesign. Binding sites in antibodies are constructedfrom the same building blocks as they are in anyprotein. The task of evolving a perfect binding siteon the antibody surface for a specific ligand iscomplementary to the task of developing aninhibitor molecule that binds in the active site ofan enzyme. Both rely heavily on our knowledge ofmolecular recognition, the mechanisms throughwhich highly specific and tight association ofproteins and ligands is achieved.

On being exposed to an antigen, the immunesystem screens a wide variety of possible bindingsites in order to optimize binding affinity. It is wellestablished in the case of large epitopes that therecognition can be achieved in multiple ways.6

Different antibodies either recognize different epi-topes7–10 or have quite different paratopes withshuffled interactions.11,12 However, in either casethe large size of the antigen appears to be crucial.But what happens if the size of an antigen isdramatically reduced, leaving very little room for

Table 1. Data collection and refinement statistics

A. Data collectionSpace group P3121Unit cell (a, b, c) (A) 75.4, 75.4, 150.8Resolution range (A) 30–1.70No. of observations 348,644No. of unique reflections 54,352Rmerge (%) 12.5Average I/sI

a 13.1 (2.0)Completeness, overall (%) 98.1 (97.9)

B. Refinement statisticsResolution range [F/s(F)O0] (A) 30.0–1.70Number of reflections 54,347Rfactor (%) 21.8 (29.7)Rfree (1277 reflections; %) 24.9 (35.4)No. of protein atoms 3454No. of cocaine atoms 22No. of water molecules 334

C. Average B valuesAntibody/waters/cocaine (A2) 29.1/38.1/31.3

D. Ramachandran statisticsMost favoured (%) 90.5Additional allowed (%) 8.7Generously allowed (%) 0.5Disallowed (%) 0.3b

E. RMSD from idealityBond lengths (A) 0.006Bond angles (deg.) 1.4Improper angles (deg.) 0.82Dihedral angles (deg.) 26.8

a Values in parentheses correspond to the outer resolutionshell (1.76–1.70 A).

b ValL51 is located in a g-turn observed in most antibodies.18

Anti-cocaine Antibody Structure 571

different modes of recognition? Will this result in aunique binding mode, either in terms of sequenceor the three-dimensional structure of the paratope?Answering these questions leads us well beyondthe scope of structural immunology and providesinsight into how small ligand recognition sites canbe built into proteins in general.

In this work we are comparing binding ofthe same small ligand, cocaine (Mrw300 Da), withsimilar affinities to two unrelated antibodies. Theseantibodies are: GNC92H2 (KdZ4.0!10K7 M; itsstructure was published recently13) and M82G2(KdZ1.4!10K7 M; this work). We discuss how twocompletely different binding sites are built upon thesame immunoglobulin scaffold and how a similaraffinity is designed into these quite differentbinding sites. We find that the two anti-cocaineantibodies show a much higher degree of structuraldiversity in ligand binding than two antibodiesagainst the potent sweetener NC174, reported byGuddat et al.14,15 The NC174 hapten binds to twodifferent antibodies in the same orientation and theantigen binding site has similar topology andlocation. In addition, the two anti-sweetener anti-bodies utilize a similar set of interactions to bind theligand, including stacking interactions, salt bridgesand hydrogen bonding. The conclusions drawnfrom our comparison of structural features of thetwo anti-cocaine antibody complexes is that one ofthem (GNC92H2) relies more on overall comple-mentarity to the ligand (both shape and electro-static), while the other (M82G2) utilizes a morespecific set of interactions, including water-mediated hydrogen bonding and stacking inter-actions. At the same time, both antibody complexesshare certain features, including cation–p inter-actions between the ligand and aromatic side-chains of the protein. This diversity of bindingmodes is directly related to the dramatic differencein topology of the antigen binding sites that appearas a deep pocket in GNC92H2 versus a groove inM82G2. Our findings thus indicate that, even for asmall antigen, highly specific recognition can beachieved in a variety of ways.

Results and Discussion

Structure of the M82G2 Fab fragment–cocainecomplex

The structure of the murine antibody (IgG1/k)M82G2 Fab fragment complexed with cocaine hasbeen determined at 1.7 A resolution as described inMaterials and Methods. The data collection andrefinement statistics are shown in Table 1.

The overall structure of the M82G2 Fab fragmentis composed of four immunoglobulin folddomains (each containing two antiparallel pleatedb-sheets; the N-terminal domains of the heavy andlight chain are called VH and VL) with thebinding site located at the tip formed by six CDR(complementarity-determining region) loops (see

Figure 2(a), below). These loops correspond to sixhypervariable sequence regions, which were namedaccording to their appearance in immunoglobulinsequences,16 as CDR L1, L2, L3, H1, H2, and H3. Wefollow here the Kabat numbering scheme, in whichthe shortest possible immunoglobulin sequence isnumbered sequentially and any insertions withrespect to that sequence are denoted with letters(see Figure 1).Five CDR loops are involved in forming the

binding site (see Figure 2(c)), with CDR L2 provid-ing no direct contacts to the ligand (the closestapproach to the ligand in this loop is the side-chainof ArgL50, which is about 6.0 A away). There is nodirect interaction between residues in CDR H2 andthe ligand; however, GluH50 is involved in a water-mediated interaction. Surprisingly, the ligand is notcompletely encased by the protein as one mightexpect for the small antigen. Rather, the binding siteis a groove between VH and VL domains.The CDR H3 loop is more disordered than the

rest of the antigen-combining site as can be seenfrom higher average B-factors for this loop(w45 A2). The most mobile region includes residues97–100 in the heavy chain. In fact, this loop is foundin three distinct conformations in cocaine-bound,native, and benzoylecgonine-bound forms of theprotein (the latter two structures are deposited inthe RCSB Protein Data Bank with codes 1RFD and1QYG).There is a lattice contact on the heavy chain side

of the binding site formed with the H179–H184 loop

Figure 1. Sequence alignment of the variable domain sequences of antibodies M82G2 and GNC92H2. Identicalresidues are shadowed. Crosses adjacent to the sequences designate the residues involved in interactions with ligand.Numbering according to the Kabat numbering scheme. Insertions are designated from the number at which an insertionoccurs and coded by single letter. These are shown at positions L27, L95, H52, H82 and H100.

572 Anti-cocaine Antibody Structure

of the constant domain of a symmetry relatedmolecule in the crystal. The only possibility ofhydrogen bonding at this crystal contact is betweenthe side-chain of AspH181 of the symmetry relatedmolecule and the backbone oxygen atom ofLeuH98, and the latter residue does not makecontact with the ligand.

Two different binding sites built upon the samescaffold

CDR comparison

Sequence alignment for the M82G2 andGNC92H2 variable domains is shown in Figure 1.Overall, the light chains are 60% identical and have81% similarity, whereas the heavy chains are only42% identical but have 70% similarity. The struc-tural alignment (Figure 2(a)) clearly indicates thatdifferences only appear in the regions with irregularsecondary structure. Obviously, these two anti-bodies have perfectly overlapping scaffolds andany structural differences are located in the loopregions, which are, however, not limited to theCDRs. The canonical structure assignments17,18 andligand-interacting residues for the loops (seeFigure 2(c) and (d)) are summarized in Table 2and discussed below in more detail.

CDR L1 in M82G2 is one residue longer than itscounterpart in GNC92H2 and the loops belong to

different canonical classes (see Table 2). TyrL32 inboth proteins interacts with the ligand via van derWaals contacts, is similarly positioned relative tothe scaffold and has the same side-chain confor-mation. HisL34 and PheL36 are part of the bindingsite in GNC92H2 but not in M82G2 due to thedifferent position of the ligand in the latter.

CDR L2 in both antibodies is in a structurallyconserved g-hairpin conformation. In M82G2, thisloop does not interact with the ligand at all, whereasin GNC92H2 TyrL49 and LeuL50 participate in vander Waals contacts.

CDR L3 in M82G2 is one residue shorter than itscounterpart in GNC92H2 and exhibits much largerconformational differences between the two pro-teins than the two other light chain CDR loops, withthe GluL93 residues being more than 10 A apart in astructural superposition of the two models (seeFigure 2(a)). In M82G2 the canonical structure 1 isadopted, whereas GNC92H2 has an unusual con-formation for this loop. In both cases the residues ofthe loop interact extensively with the ligand, withGNC92H2 having relatively more backbone inter-actions. Also, in M82G2 this loop interacts pre-dominantly with the tropane moiety of the ligand,whereas in GNC92H2 this loop interacts with boththe tropane and benzoyl groups.

Despite the high degree of sequence similarity,the CDR H1 0s adopt quite different conformationsin the two proteins. M82G2 has this loop in

Figure 2. (a) The structural alignment of the variable domains of antibodies M82G2 and GNC92H2. M82G2 is cyan(VL) and green (VH) and the CDRs are blue and dark green for the respective chains. GNC92H2 is yellow (VL) andmagenta (VH) and its CDRs are orange and red, respectively. The cocaine ligands bound to M82G2 (green) andGNC92H2 (pink) are also shown. (b) The cocaine ligand (yellow) bound to M82G2 is shown with an electron densitymap with coefficients (2foKfc) contoured at 1.5s. The elements of specific protein–ligand interactions are shown: (i) thebinding site water molecule (red); (ii) the cation–p interaction with TyrL32 (green); (iii) the stacking interaction withTrpH33 (cyan). (c) The cocaine-binding site of M82G2. Only the residues forming contacts with the ligand are shown.(d) The cocaine-binding site of GNC92H2. Only the residues forming contacts with the ligand are shown.


canonical structure 1, whereas GNC92H2 has anunusual conformation. Interestingly, the sequenceof the loop itself does not prevent canonicalstructure 1 in GNC92H2, as one can see fromcomparison to the carbohydrate-binding murineantibody Se155-4.19 Moreover, forcing the loop intocanonical structure 1 would not disrupt the bindingsite (the loop barely interacts with ligand). The loopis somewhat disordered in GNC92H2 and formspart of a lattice contact, and this intermolecularinteraction may cause the unusual conformation.

The H1 loop in M82G2 contains the veryimportant residue TrpH33 that alone providesabout 49% of all the protein–ligand contacts and isinvolved in a stacking interaction with the phenyl

ring of cocaine. In contrast, the loop in GNC92H2comprises a tiny part of the binding site, giving riseto a single contact through TyrH32.The H2 loops are of different length in M82G2

and GNC92H2 and adopt different conformations(4 and 2b, respectively; see Table 4). They do notform any direct contacts with the ligand.The H3 loops have bulged and non-bulged torso

conformations20 in M82G2 and GNC92H2,respectively. The head region in M82G2 resemblesthat of an anti-myohemerythrin antibody B13I2,21

which represents an alternative conformation forthis 15 residue loop.20 However, the loop has highmobility and hence the assignment is not firm. Inboth proteins the loop provides a significant part of

Table 2. Comparison of structures of CDR loops in M82G2 and GNC92H2

CDRloop M82G2 GNC92H2

L1 5 residue insertion at position 27 4 residue insertion at position 27Canonical structure 4 Canonical structure 5TyrL32 (7), HisL27D (1) HisL34 (12), TyrL36 (3), TyrL32 (2)

L2 g-Hairpin g-HairpinLeuL50 (4), TyrL49 (1)

L3 Insertion at position 95 (Pro–Pro)Canonical structure 1 Unusual structureHisL91 (14), TyrL94 (2), PheL96 (2), LeuL92 (1) GluL93 (8), ArgL92 (6), SerL91 (4), LeuL89 (2), TrpL96 (2)

H1 Canonical structure 1 Unusual structureTrpH33 (47), AspH31 (2) TyrH32 (2)

H2 3 residue insertion at position 52 1 residue insertion at position 52Canonical structure 4 Canonical structure 2bAsnH52A (1)

H3 2 residue insertion at position 100Bulged torso Non-bulged torsoValH95 (9), GlnH97 (4), ProH96 (3), GlyH99 (1) TyrH95 (15), ProH98 (7), AspH101 (4), SerH97 (2), GluH93 (1)

Listed residues are those that form contacts with the cocaine ligand. They are ordered by the number of 4.1 A contacts, given inparentheses. Canonical structures were assigned according to Al-Lazikani et al.18 & Morea et al.20

Table 3. Comparison of the size and polarity of the surface buried upon binding in M82G2 and GNC92H2

Buried surface (A2) M82G2 GNC92H2

Ligand, non-polar/polar 158/24 221/33Protein, non-polar/polar 177/101 242/86Total, non-polar/polar 335/125 463/119

Displaced water moleculesProtein, overall 19.7G0.2 11.3G0.1Protein, surface 12.7G0.2 8.9G0.1Ligand, surface 10.4G0.2 14.1G0.1Total, surface 23.1G0.3 23.0G0.2

Molecular surface calculations were done using Surface Racer.40 counts of displaced water molecules are given with standard errors.The calculation of the total number of water molecules on the surface of a free cocaine molecule in solution gives 18.1G0.1.

Table 4. Comparison of the protein–ligand interactions in M82G2 and GNC92H2

Interaction M82G2 GNC92H2 DGGNC2H2–DGM82G2 (kcal/mol)

Hydrophobic: buried non-polar surface (A2) 335 463Water molecules released w23 w23 w0

van der Waals: contacts 94 75 C2H-bonding: water-mediated hydrogen bonds 1 1 w0Electrostatic: distance between charged groupsa 7.6 A 4.9 A K331

Cation–p: tropane nitrogen–tyrosine distanceb 4.5 A 4.3 A w0Stacking (p–p) of benzoyl ring TrpH33 Absent C130

a Distance from the tropane nitrogen atom to the nearby anionic side-chain (GluH50 in M82G2 and AspH101 in GNC92H2).b Distance from the tropane nitrogen atom to the center of the aromatic ring.


the ligand-complementary surface. Also, inGNC92H2 TyrH95 provides a cation–p interactionwith the tropane nitrogen atom of cocaine.

Ligand orientation

The two antibodies bind the cocaine ligand indramatically different modes (see Figure 3). InM82G2, the binding site is a groove and one sideof the ligand remains exposed to solvent uponbinding. In GNC92H2 the cocaine ligand is almostcompletely buried with the tip of its benzoate

moiety facing solvent. Also, as we discuss below inmore detail, there are significant differences in howthe affinity is built into the two different structures.In general, M82G2 interacts more specifically withthe ligand whereas GNC92H2 appears to rely onoverall surface complementarity.

The molecular structures of the cocaine ligandsbound to the two antibodies are very close (r.m.s.d.value in atomic positions between the two struc-tures is 0.32 A). In the case of M82G2 there is anadditional rotation of the benzoyl ring, apparentlydue to p–p interaction with TrpH33, resulting in

Figure 3. Comparison of the topology of the bindingsites of antibodies M82G2 ((a), ligand in pink) andGNC92H2 ((b), ligand in green). The molecular surfacesand corresponding minimum distances from the ligandswere calculated using GRASP41 and rendered usingPovScriptC.42 Surfaces are colored based on the mini-mum distance to the ligand to emphasize the binding site.The ligand binding site for M82G2 appears more as agroove and that for GNC92H2 more like a deep pocket.Both binding sites are viewed from the same anglerelative to the immunoglobulin scaffold.

Figure 4. Comparison of the conformation of thecocaine ligand bound to M82G2 (yellow), GNC92H2(green) and in free form43 (magenta). The position of theTrpH33 of the antibodyM82G2 is shown. (a) Note that thebenzoyl ring in M82G2 is not co-planar with the carboxylmoiety of the benzoate. (b) Note the conformationalrearrangement of the methyl ester in both antibody-bound forms of the ligand.


non-coplanarity of the carboxylate moiety of thebenzoate group (see Figure 4(a)). In both structuresthere is also a notable loss of planarity of the methylester group (see Figure 4(b)), the structural origin ofwhich is not immediately clear.

These two antibodies were in fact raised againstdifferent haptens, as shown in Figure 5. However,

both antibodies appear to be able to bind bothhaptens and thus their structural differences couldnot be directly linked to the discrepancies betweenthe procedures used to generate them. We wouldrather suggest that there are multiple ways to buildthe binding site for a given ligand on the sameprotein scaffold (even if the ligand is relativelysmall and therefore provides a limited number ofepitopes) and the immune system randomly picks aspecific solution to the recognition problem. Thisconclusion is also important in the broader contextof protein–ligand interactions, indicating thatsmall-molecule recognition can be achieved indiverse ways.

Similar affinity is designed into quite differentstructures

Hydrophobic/dispersive interactions

The overall topology of the two binding sites isobviously different with the result that less of thenon-polar solvent-accessible ligand surface isburied in the M82G2 complex (by about 63 A2)than in the GNC92H2 complex. The binding sitesurface is also less hydrophobic in the case ofM82G2, as seen in Table 3, and this brings theoverall non-polar buried surface area differencebetween two antibody–antigen complexes toabout 128 A2. Assuming a transfer energy22 of20–25 cal/mol per A2, this difference may account

Figure 5.Chemical structure of cocaine and the haptensthat were used to produce antibodies M82G2 andGNC92H2. BTG is bovine testes b-galactosidase.


for a relative loss of about 2–3 kcal/mol of bindingfree energy by M82G2.

When compared to other antibodies, GNC92H2buries an unusually large portion of its ligand. Weanalyzed 65 different antibody–hapten structuresfrom the Protein Data Bank23 and only three of themencase a larger ratio of the accessible surface of their

respective ligands. Moreover, when the set isnarrowed to 16 structures for which the accessiblesurface area of a free ligand is not more than 60 A2

different from that of cocaine, GNC92H2 turns outto be the only antibody that buries more than 80% ofits ligand. M82G2, on the contrary, covers less of itsligand’s surface than antibodies do on average, asone can see in Figure 6. Consequently, theGNC92H2 binding site can be described as relyingmore on surface complementarity, whereas theM82G2 binding site is more polar and thus involvesmore specific interactions.

The above estimate of the extra binding freeenergy assigned to the more hydrophobic bindingsite in GNC92H2 is based on a simplifyingassumption that the strength of hydrophobicinteractions is proportional to the buried surfacearea. This assumption may become incorrect for asmall concave binding site. Hydrophobic inter-actions are due to water molecules displaced froma binding site by a ligand, and this contribution maybe substantially reduced when the cleft is toonarrow to allow hydration. Indeed, the results ofthe molecular dynamics simulation (see Table 3)indicate that while more waters are displaced fromthe surface of the ligand by GNC92H2, there are lesswater molecules in its pocket-like binding site.Somewhat paradoxically, the smaller surface area ofthe shallow binding site of M82G2 may in factprovide more stabilizing free energy in the form ofhydrophobic interactions.

van der Waals interactions

In the M82G2–cocaine complex about 60% of thesurface of the ligand is covered, whereas over 85%of the ligand surface is covered in the GNC92H2–cocaine complex. Nevertheless, the total number ofprotein–ligand van der Waals contacts is 94 for theM82G2 and 75 for the GNC92H2. Assuming that a

Figure 6. The fraction of molecu-lar surface area buried upon inter-action of a ligand with an antibodyversus the molecular surface area ofthe free ligand. The data represent65 structures from the RCSBProtein Data Bank, all of which areantibodies complexed with non-peptide antigens. Black dots rep-resent the antibodies discussedhere, M82G2 (lower black dot) andGNC92H2 (upper black dot).


single contact contributes about 0.1 kcal/mol to thefree energy of binding, this would provide about2 kcal/mol more for the M82G2–cocaine inter-action.

The larger number of contacts for the M82G2 isexclusively due to the stacking of the benzoatemoiety of the cocaine ligand against the indole ringof the TrpH33. The number of van der Waalscontacts formed by the ecgonine part of the ligandis 34 and 47 in M82G2 and GNC92H2, respectively.For the benzoate moiety these numbers are 60 and28, and TrpH33 of M82G2 provides over two-thirdsof these contacts.

Figure 7. Protein–ligand interactions in the cocaine-binding site of M82G2. (a) Schematic diagram of theM82G2 cocaine binding site. (b) The putative hydrogen-bonding network holding the binding site watermolecule.

Hydrogen bonding

In both structures the cocaine molecule is boundin the energetically favorable chair conformationand thus has an intramolecular hydrogen bondbetween the carbonyl group of the methyl ester andthe tropane nitrogen atom (see Figure 2(b)). InGNC92H2, the hydrogen is shared by a nearbywater molecule that forms another hydrogen bondto the backbone carbonyl group of SerH97. M82G2lacks this interaction (which could contribute1–2 kcal/mol to the binding energy24), but it has acompensating water-mediated interaction with thebenzoate carbonyl oxygen atom that we willdescribe below.

The binding site water molecule in M82G2 ispart of a putative hydrogen-bonding networkthat involves six residues and the ligand (seeFigure 7). The water oxygen atom is 3.0 A fromthe cocaine benzoate carbonyl oxygen, which mayserve as a hydrogen bond acceptor. The water isalso at 2.8 A from the N3 nitrogen of HisL91, andforms a hydrogen bond with the GluH50 3-oxygenwith the donor–acceptor distance of 2.7 A. Furtheranalysis of the hydrogen bonding network, whichis described in more detail below, leads to theconclusion that the binding site water molecule isneutral (neither hydronium, nor hydroxide ion) andAspH35, the second anionic residue in the bindingsite, is protonated and therefore also neutral.

The HisL91 Nd forms a hydrogen bond to N3 ofHisL34. Two 2.6 A long hydrogen bonds are formedbetween the 3- and d-oxygen atoms of GluH50 andAspH35 and between GluH50 and TyrL94. It isimportant to emphasize for the later discussion ofthe electrostatic interactions in the binding site thatthis necessitates at least one of the two (AspH35and/or GluH50) carboxylate side-chains to beprotonated. This does not contradict the fact thatthe pH in the experiment (7.2) was higher thanthe normal pKa value of these groups, since theirproximity to each other and electrostatic repulsionbetween them should accordingly promote theprotonation of at least one. We believe that it isAspH35 that is protonated because the 3-oxygenatom of GluH50 is about 0.7 A above the plane ofthe AspH35 carboxylate. The binding site watermolecule is, however, about 0.7 A above the planeof the imidazole ring of HisL91, and angles formed

by the cocaine benzoate carbonyl oxygen atom (O2),the water molecule, and the HisL91 N3 nitrogen and3-oxygen of GluH50 are w1098 and w1088, respect-ively. Because the (N3–O2–O3) angle is w1308, thewater molecule is not a hydronium ion (theexpected angle would then be w1138) and thushas a shared hydrogen bond to the ligand moleculeand GluH50.

Electrostatic interactions

The tropane nitrogen atom of the cocainemolecule is protonated at physiological pH(pKaw8.6) and therefore the ligand possesses anoverall positive charge. As the authors of the


structural study of GNC92H213 have pointed out,two residues at the bottom of the binding pocket,GluH93 and AspH101, may provide electrostaticcomplementarity. We must emphasize that, just likethe AspH35–GluH50 pair in M82G2, these tworesidues are also likely to form a hydrogen bondwith each other and therefore probably only one ofthem is charged.

Besides M82G2 and GNC92H2, there are threeother instances of Glu–Glu or Glu–Asp pairspositioned within hydrogen bonding distance inactive sites of antibodies specific to small ligands.In one of them, the OXY-COPE catalytic antibody,25

GluH35 and GluH50 are involved in an interactionsimilar to one found in M82G2, holding the watermolecule that is also hydrogen-bonded to the ligandand to TyrL96. Interestingly, an affinity-maturedOXY-COPE catalytic antibody26 still contains theGlu–Glu pair but the water molecule loses itshydrogen bond to the ligand.

Two other instances, an anti-sweetener anti-body14 and an anti-PCP antibody,27 resemble thecase of GNC92H2. The GluH35–GluH50 andGluL34–AspH99 pairs in these antibodies do notform direct hydrogen bonds with any watermolecules but are in both cases located in thevicinity of the cationic groups of the ligands. Theanti-morphine antibody 9B128 provides anotherexample of the GluH35–GluH50 pair, but in thiscase GluH50 simultaneously forms a salt bridge tothe cationic ligand, similar to many instances of saltbridges with arginine and lysine side-chains inantibodies specific to various protein antigens.

In summary, the structurally similar arrangementof two anionic side-chains in M82G2 and GNC92H2may serve different purposes, holding an importantwater molecule in one case and providing electro-static complementarity in the other. It is alsopossible that in M82G2 these residues represent aremnant from early stages of affinity maturation,when one of them could have served to form a saltbridge with the cationic cocaine ligand.

Cation–p interactions

A cationic ligand, such as cocaine, may beinvolved in cation–p interactions with antibodyaromatic side-chains and this may increase itsbinding affinity by 2–5 kcal/mol.29 Indeed, in ourstructure of the M82G2–cocaine complex, thetropane nitrogen atom is properly positioned nextto TyrL32. A similar interaction is found inGNC92H2, where the tropane nitrogen atom inter-acts with the p-cloud of the aromatic ring ofTyrH95.

Stacking interactions

TrpH33 is perhaps the most important interactingresidue in the cocaine–M82G2 complex. It buries asignificant part of the ligand, alone providing abouthalf of the contact surface. Moreover, it is stackedagainst the phenyl ring of the cocaine benzoate

moiety (see Figure 2(b)). Such a stacking interactionmay contribute up to 1 kcal/mol to the bindingenergy30 due to favorable electrostatic interactionbetween two aromatic p-clouds. Also, such anarrangement of two aromatic rings contributessignificantly to van der Waals interactions.

Structural basis of binding affinity

The above analysis of the different interactionsinvolved in the binding of the cocaine molecule totwo distinctly different binding sites can now besummarized in order to address the question ofhow similar affinity is achieved by them.

Two kinds of interactions present in bothstructures are likely to contribute similarly tobinding affinity. They are: a single water-mediatedhydrogen-bonding interaction found in both com-plexes and a cation–p interaction between thetropane nitrogen atom of cocaine and a nearbytyrosine residue. It is important to note that in eachcase the interactions are with different parts of theantibody.

One interesting conclusion drawn from ouranalysis of the two binding sites is that hydrophobicinteractions are likely to contribute similarly to theirrespective binding affinities. The drastic differencein the overall topology of binding sites (pocketversus groove) allows GNC92H2 to bury a largerpart of the ligand, yet the same number of waters isdisplaced from protein and ligand surfaces in bothcases. It appears that for a ligand of this size theadvantage of pocket-like binding site in releasingmore water molecules from the ligand surface iscompensated by the fact that the binding site itselfis somewhat dehydrated because of its highcurvature.

Electrostatic interactions are likely to providelarger contributions to the stabilization of theantibody–antigen complex in the case ofGNC92H2. As discussed above, the “electrostaticcomplementarity” of the GNC92H2 site hinges onthe protonation states of the CDR polar residues.But in any case, the tropane nitrogen atom that isthe cation in the cocaine molecule is definitely at alarger distance from anionic side-chains in M82G2and thus interacts less strongly with them. Assum-ing an effective dielectric constant of 8,31 weestimate the difference in electrostatic stabilizationof a liganded form of these two proteins asw3 kcal/mol.

This energetic deficiency due to electrostaticinteractions in the M82G2–cocaine complex iscompensated by a stacking interaction betweenthe cocaine phenyl ring and TrpH33, the interactionlacking in the GNC92H2 complex. This compen-sation is twofold, directly from the electrostaticinteraction between p–electron clouds and fromimproved van der Waals interactions betweenprotein and benzoate moiety of the cocaine ligand.Hence, the conclusion may be drawn that evensmall ligands that have a limited number of


recognition determinants can bind to structurallydiverse binding sites with comparable affinities.

Structural diversity of binding sites produced inindependent hybridoma trials with somewhatdifferent haptens (see Figure 5) is certainlyexpected. A more surprising conclusion from thestructural comparison is that the same energy ofbinding can result from different interactions, withhydrophobic–electrostatic interactions in theGNC92H2–cocaine complex being substituted by astacking interaction in theM82G2–cocaine complex.This may reflect the fact that the immune systemdoes not have to achieve the strongest possiblebinding affinity if micromolar affinities are highenough to protect against infection. Also, a smallmolecule like cocaine is perhaps not small enoughto converge recognition to a unique binding mode.

Conclusions

We report the structure of an anti-cocaineantibody M82G2 in complex with cocaine andcompare it with another anti-cocaine antibody,GNC92H2. Both proteins share the same fold, yettheir respective binding sites have strikinglydifferent topologies. As expected for a smallantigen, the GNC92H2 binding site is a deep pocketthat buries most of the ligand surface uponinteraction. The cocaine binding site of M82G2 canbe described rather as a shallow depression on theprotein surface, a topology that is considered to bemore appropriate for larger antigens such aspeptides. This change in binding site topology isdue to conformational rearrangements of CDRloops and can therefore be directly attributed totheir sequence differences.

Despite significant structural differences, the twobinding sites provide similar affinity for the cocaineligand. We have concluded that despite the abilityof the pocket-like binding site of GNC92H2 to covera larger portion of the ligand, hydrophobic inter-actions contribute similarly to the binding affinity ofboth antibodies, simply because a concave cavitythis small could not accommodate the same numberof water molecules as would a flat surface of thesame size. M82G2 also has more favorable van derWaals interactions with the cocaine ligand, mostlydue to stacking of its benzoate moiety against atryptophane side-chain. Both antibodies utilizewater-mediated hydrogen bonding and cation–pinteractions, and these should contribute equally totheir binding affinity. Both antibodies have a doubleanionic side-chain motif at the bottom of thebinding site, and better orientation of the cationiccocaine ligand allows GNC92H2 to have a morefavorable electrostatic interaction with it. In con-clusion, two antibodies achieve similar affinity indifferent ways, replacing overall shape and electro-static complementarity (GNC92H2) by stackinginteractions (M82G2).

It is well understood that protein–protein recog-nition may be achieved in diverse ways, because of

the large number of possible epitopes for largeantigens. It is also well understood that protein–protein recognition sites are big enough to providehigh affinity via various sets of interactions. It isexpected, however, that for progressively smallerligands recognition may converge to a uniquemode, because the binding site has to encompassthe entire ligand and the number of potentialantigenic determinants is reduced. We have foundthat for a ligand built of only 22 atoms a bindingmode in the 0.1 mM affinity range is not yet unique,but may be achieved using strikingly differentrecognition sites, both topologically and energeti-cally. Not only does this observation demonstrateoutstanding flexibility of the immune system inhapten recognition, but also suggests thatsmall effector molecules may be recognized bystructurally unrelated receptors.

Materials and Methods

Antibody production and Fab purification

The murine antibody M82G2 against cocaine wasproduced by hybridoma technology and provided byRoche Diagnostics, Inc. (Indianapolis, IN). Fab fragmentswere obtained by papain digestion followed by chromato-graphic purification. Briefly, 10 mg/ml antibody isincubated for at least four hours at 37 8C in the presenceof 10 mM cysteine, 1 mM EDTA and 0.1 mg/ml papain in0.1 M phosphate buffer (pH 7.2). The reaction is thenstopped by adding 20 mM iodoacetamide. Digestedantibody was applied to a gel-filtration column(Sephacryl S-200 HR from Amersham BioSciences(Piscataway, NJ)) and eluted with 10 mM Tris(pH 8.0) at4 8C. Chromatographic peaks for the Fc and Fab frag-ments overlap but the amount of protein in overlappingfractions is small and therefore no further purification isnecessary.Protein purity was checked by SDS-PAGE under non-

reducing conditions under which Fab appears as aw50 kDa fragment and Fc appears as a w25 kDafragment. Purified protein was exchanged into 10 mMTris–HCl (pH 8.0) at 4 8C, concentrated to w5 mg/ml bycentrifugation and stored at 4 8C for crystallization trials.Protein concentration was determined by measuringabsorbance at 280 nm assuming an extinction coefficientof 1.4 (mg/ml)K1 cmK1.

Crystallization and transfer to sodium malonate

M82G2 was screened for preliminary crystallizationconditions using sparse matrix screens from HamptonResearch (Laguna Niguel, CA). X-ray quality crystals ofabout 0.3 mm in size appeared after two to three weeksincubation at room temperature in the hanging drop overa reservoir containing 2 M ammonium sulfate and5%(v/v) iso-propanol. Additional screening did notimprove the quality of the crystals. Removal of iso-propanol substantially delayed crystal growth.Mature crystals are transferred stepwise into 70%

saturated sodium malonate(pH 7.2), and subsequentlysoaked overnight in sodium malonate containing 300 mMcocaine–HCl (purchased from Cerilliant (Austin, TX) as a1 mg/ml solution in methanol). Transfer to sodium


malonate is necessary because cocaine does not bind tothe protein in the original crystallization solution (noelectron density corresponding to the ligand was found inthe binding site under such conditions). Crystals werethen flash-frozen in liquid nitrogen and stored for datacollection.

Data collection and processing

Data were collected at the National SynchrotronLight Source X6A beamline on an ADSC Quantum210CCD area detector. Data were processed with DENZOand further scaled with SCALEPACK.32 See Table 1 fordetails.

Molecular replacement

Fifteen antibody structures were picked randomly fromthe RCSB Protein Data Bank to serve as models for thedetermination of phases by the method of molecularreplacement. Each one was split into constant andvariable domains to use as separate probes and theiroriginal sequence was preserved. We used AMoRe33 tosolve the structure, as described below.First, using data in the resolution range 8–3.5 A and an

integration radius of 25 A, all 30 probes (15 constant and15 variable domains) were subjected to rotation searches.As expected, constant domains gave more stablesolutions and were then used for translation searchesfollowed by rigid-body refinement. At this step the rightspace group was determined and the best solution picked(PDB code 1AR3, RZ46.0%). With the variable domains,4 out of 15 failed to produce an outstanding solution inrotation searches; the 11 successful ones were subjected totranslation searches with the constant domain fixed. Thebest solution (PDB code 1AXT, RZ50.5% when usedalone) combined well with the constant domain solutionand further lowered the starting R-factor to 37.3%.

Refinement

Positional refinement of the M82G2–cocaine complexstructure was done in CNS.34 The first step of refinementwas simulated annealing followed by a cycle of manualrebuilding and variable domain sequence adjustment,conjugate gradient minimization and grouped B-factorrefinement. At this point, clear electron density for theligand was found in the binding site and the cocainemodel was introduced. The starting model of the ligandwas built using the coordinates from the complex ofGNC92H2 with cocaine,13 adjusting two dihedral anglesdescribed by Yang et al.35 in order to fit the molecule intothe density. Subsequent refinement for the ligand wasdone in CNS34 with topology files generated by programXPLO2D,36 with no dihedral restraints applied.Difference electron density maps with coefficients

(foKfc) were inspected and water molecules wereincluded for peaks exceeding 3s and located in properpositions to form hydrogen bonds with protein. Afterpositional and B-factor refinement those water moleculeswhich produced less than 1s electron density peaks in anelectron density map with coefficients (2f0Kfc) wereexcluded from the model.The sequence of the constant domain was deduced

from the Wu & Kabat database.37 The final R-factor of21.8% for all data to 1.7 A resolution was achieved after acycle of manual rebuilding, conjugate gradient minimiz-ation and restrained individual B-factor refinement for

the complete model (Rfreew24.9%). The bulk solventcorrection implemented in CNS was modified so that nointernal cavities were included with the mask.

Molecular dynamics simulation

Molecular dynamics simulations at 300 K were per-formed using GROMACS.38,39 Crystal structures of thecocaine-bound forms of M82G2 and GNC92H2 (PDBcodes 1Q72 and 1I7Z) were reduced to the variabledomains Fv only (residues past L108/H113 and thecocaine ligand were removed). Crystallographic watermolecules found within 3.6 A from heavy atoms in Fvwere included in the starting model which was sub-sequently solvated in aw70 A box (the size was chosen toallow at least 5 A from the starting model to the edge ofthe box) with periodic boundary conditions. Theunliganded form models included 6460 and 6139 watermolecules for M82G2 and GNC92H2, respectively. Thesolvated models were subjected to gradient minimizationin order to regularize water molecules, followed by a10 ps preliminary molecular dynamics run. The finalstructure was used as the starting point for a longer(100 ps) simulation, the result of which was used foranalysis.The cocaine ligand was modeled with a cationic charge

placed at the tropane nitrogen atom. The cocaine-boundforms were generated essentially in the same way as thenative ones (containing 5788 and 5576 water moleculesfor the M82G2 and GNC92H2 complexes, respectively).A separate simulation was performed for the cocainemolecule alone in a smaller box containing 637 watermolecules.To determine the number of water molecules displaced

by the ligand, we used the following procedure. Snap-shots of the unliganded structure were superimposed onthe crystal structure of the liganded form. All the watermolecules within 3.6 A from any cocaine heavy atomwere counted as displaced by the ligand. Additionally,the subset of displaced water molecules that wereoriginally within 3.6 A from any heavy atom in theprotein was determined and these were counted aswater molecules displaced from the surface. To determinethe number of water molecules displaced from theligand surface the total number of water moleculeswithin 3.6 A from the cocaine molecule in the ligandedform was compared to the same number obtained forthe free cocaine molecule. Snapshots were taken every0.5 ps and counts of displaced water molecules wereaveraged.

Coordinates

The atomic coordinates and structure factors for thecomplex of the Fab M82G2 with cocaine have beendeposited in the RCSB Protein Data Bank with accessioncode 1Q72.

Acknowledgements

This work was supported by a grant from RocheDiagnostics, Inc. and by NIH grant GM26788 toG.A.P. & D.R.


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Edited by I. Wilson

(Received 23 February 2005; accepted 17 March 2005)

Diversity in Hapten Recognition: Structural Study of an Anti-cocaine Antibody M82G2

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