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Conformational ChangesAntibodies Reveals Binding-Related A Systematic Comparison of Free and Bound

Inbal Sela-Culang, Shahar Alon and Yanay Ofran

ol.1201493http://www.jimmunol.org/content/early/2012/10/12/jimmun

published online 12 October 2012J Immunol 

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The Journal of Immunology

A Systematic Comparison of Free and Bound AntibodiesReveals Binding-Related Conformational Changes

Inbal Sela-Culang, Shahar Alon, and Yanay Ofran

To study structural changes that occur in Abs uponAg binding, we systematically compared free and bound structures of all 141 crystal

structures of the 49 Abs that were solved in these two forms.We found that many structural changes occur far from the Ag binding site.

Some of them may constitute a mechanism for the recently suggested allosteric effects in Abs. Within the binding site itself, CDR-H3 is

the only element that shows significant binding-related conformational changes; however, this occurs in only one third of the Abs.

Beyond the binding site, Ag binding is associated with changes in the relative orientation of the H and L chains in both the variable and

constant domains. An even larger change occurs in the elbow angle between the variable and the constant domains, and it is significantly

larger for binding of big Ags than for binding of small ones. The most consistent and substantial conformational changes occur in

a loop in the H chain constant domain. This loop is implicated in the interaction between the H and L chains, is often intrinsically

disordered, and is involved in complement binding. Hence, we suggest that it may have a role in Ab function. These findings provide

structural insight into the recently proposed allosteric effects in Abs. The Journal of Immunology, 2012, 189: 000–000.

The inherent flexibility of proteins is essential for theirfunction, allowing them to adopt new conformations and,in turn, bind to distinct ligands (1–3). Experimental and

structural studies suggest that Abs are no different, showing someflexibility upon Ag binding (4). This flexibility was suggested tobe essential to their ability to bind multiple Ags (3). However,although the existence of conformational changes in the Ag bindingsite has been widely recognized, their role in the adaptive immunesystem has not been structurally elucidated. Furthermore, under-standing these conformational changes should help to improve Abmodeling, docking, and engineering (5–9).Until recently, the variable and constant domains were consid-

ered functionally independent, where Ab affinity and specificity aredetermined by the variable domains, and Ab isotype and effectorfunction are mediated by the constant domains (10, 11). Thus, mostof the Ag binding-related structural changes were expected to occurat the variable domains. However, recent reports provide someevidence for binding-related allosteric effects in Abs. It wasdemonstrated that Ag binding, as well as the structure of thevariable domains, may be influenced by changes in the constantdomains (11–24). For example, Pritsch et al. (13) demonstratedthat two human mAbs sharing identical variable domains, butexpressing different isotypes, bind tubulin with significantlydifferent affinities. Because the differences observed were found atthe Fab level, the investigators suggested a role for the constant

heavy-1 (CH1) domain in shaping the Ag binding site. Janda andCasadevall (11) used circular dichroism spectra to analyze fourmAb isotypes of the 3E5 family that share identical variable do-mains. They found that the different isotypes undergo differentstructural changes upon binding to a common Ag, providing evi-dence for structural cross-talk between the constant and the vari-able regions. Tudor et al. (24) even suggested that the epitopesrecognized by two anti–HIV-1 IgG1 and IgA2 Abs with identicalvariable domains are only partially overlapping. Furthermore,several studies suggested that long-distance structural changes maybe transferred in the other direction as well, from the variable tothe constant domain, potentially influencing effector activation(10, 25–27). For example, Oda et al. (25) showed that the bindingof staphylococcal protein A (SPA) or streptococcal protein G tothe constant domain was inhibited by hapten binding in severalmAbs. Results of isothermal titration calorimetry also revealed thatthe Ka for the interaction of SPAwith IgG2b was decreased by theaddition of hapten. Because SPA and streptococcal protein G areknown to bind to IgG CH domains, such as CH1, CH2, and CH3,the investigators concluded that signals resulting from the haptenbinding induced a conformational change at these constant domains.A different example was provided by Horgan et al. (27), who ob-served differences in complement activation of two Abs that differonly in their variable-heavy (VH) domain. A possible mechanismfor such observations was suggested by Torres and Casadevall (10),in which electrostatic and hydrophobic interactions resultingfrom differences in the microenvironment of CH domains (e.g.,pH, ionic strength) may affect the Ag binding site. Additionally,the arrangement of the Fab constant domains relative to the vari-able domains and to each other may increase the probability of anappropriate VH–variable light (VL) relative orientation (28),which, in turn, can shape the Ag binding site (4, 29, 30).Early analyses during the 1990s and the beginning of the mil-

lennium attempted to characterize structural changes that occur inAbs upon Ag binding (8, 28–35). The results of these studies, whichtypically relied on only a few Abs, revealed a large variation in thebinding-related conformational changes in individual Abs. Thisincluded reports of very small overall changes, side-chain move-ments, large rearrangement of one or more of the CDRs, change inthe VH–VL relative orientation, and, in some cases, combinations

Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel

Received for publication May 30, 2012. Accepted for publication September 17,2012.

This work was supported by Grant 511/10 from the Israeli Science Foundation.

Address correspondence and reprint requests to Dr. Yanay Ofran, Goodman Facultyof Life Sciences, Nanotechnology Building, Bar Ilan University, Ramat Gan 52900,Israel. E-mail address: yanay@ofranlab.org

The online version of this article contains supplemental material.

Abbreviations used in this article: CE, combinatorial extension; CH1, constant heavy-1;CH1-1, first loop of the constant heavy-1 domain; CL, constant light; FR, framework;MSTA, multiple structure alignment; PDB, Protein Data Bank; RMSD, root-mean-square deviation; SPA, staphylococcal protein A; S-S, disulfide; VH, variable heavy;VL, variable light.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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of some or all of the above. Recent studies used newer structure-comparison methods (4, 36) and used the growing number of avail-able structures (37–40) to perform comparative research to identifynew and more general characteristics of Ab structures. However,many of these studies did not compare free and bound structuresof the same Ab (4, 37, 40), focused only on the VH–VL relativeorientation (4, 37, 38, 40), or included only a limited number ofstructures (4, 36, 39). None of these studies elaborated on the Fabconstant domains.In this study, we describe a comprehensive systematic structural

comparison of all Abswhose crystal structure has been experimentallydetermined both in free and in Ag-bound forms. We characterizechanges at the residue level in both the variable and the constantdomains, as well as changes in the heavy–light and variable–constantrelative orientations. We also compare different bound structures ofthe same Ab to determine whether the observed changes could beconclusively ascribed to Ag binding rather than to intrinsic flexibility.

Materials and MethodsDataset

The Protein Data Bank (PDB) ID of all crystal structures containing Abs, aswell as the Ab chain IDs in these PDB files, were identified using Inter-national Immunogenetics Information System/3Dstructure-DB, version4.5.0 (41, 42). For each PDB file, only one biological unit (the first one thatappears in “REMARK 350”) was considered. Heavy and light labels wereassigned to the Ab chains by running BLAST (43) against an examplesequence of one H chain and one L chain and selecting the one with thehigher E value. The Ab in each structure was labeled either “free” (if noother chain in the same biological unit exists within 6 A of the Ab variabledomain) or “bound” (otherwise). Structures containing an Fc region,structures of germline Abs, and scFv structures were ignored. We alsodiscarded structures of Abs bound to a non-amino acid Ag, to a peptideAg with less than five amino acids, or to an Ag that is an Ab itself. Toidentify different crystal structures of the same Ab, the sequences of all Abchains were clustered using BLASTCLUST (44) (performed separately forH and L chains), requiring 100% sequence identity and 95% coverage.Coverage was required in only one sequence, so that two structures of thesame Ab, one containing both variable and constant domains and the othercontaining only the variable domain, would be grouped together. Twostructures were considered as being of the same Ab if both of their chains(heavy and light) shared 100% sequence identity. The final dataset con-tained only Abs that were found to have at least one free and one boundstructure. The PDB IDs of the Abs in this dataset are shown in Table I. Forexample, cluster number 7 contains one free structure and six bound ones.Thus, for this Ab, we measured free–bound changes over the 6 free–boundpairs and bound–bound changes over the 15 bound–bound pairs.

Position-based comparison

The analysis described below was performed separately for the L and Hchains. Structural features for each residue of each protein in the datasetwere compared between each pair of structures (i.e., between one free andone bound structure or between two bound structures) of the same Ab. Thestructural features were calculated as follows:

1) Solvent accessibility was calculated with STRIDE (45) and DSSP(46). For bound structures, the Ag was not included in the calcula-tion so as to avoid changes that are a direct result of Ag bindingrather than a conformational change. The change in solvent accessi-bility was then calculated as the absolute value of the differencebetween the solvent accessibility of the two structures. Positions forwhich this change was not consistent between DSSP and STRIDEwere ignored.

2) The number of interchain contacts was calculated for each residue bycounting the number of amino acids that it contacts in the oppositechain. Two amino acids are considered to be in contact if any of theirrespective atoms are within 6 A of each other. The change in inter-chain contacts was calculated as the absolute value of the differencebetween the number of interchain contacts of the two structures.

3) Ca and heavy atoms root-mean-square deviation (RMSD) of twocompared structures were calculated for each position, followingtheir structure alignment using combinatorial extension (CE) (47).The structure alignment was performed separately for the constantand variable domains.

Next, corresponding positions in different Abs were identified using thenomenclature of the Multiple Structure Alignment (MSTA) of 96 H chainsand 98 L chains described previously (48). For structures that were notincluded in the MSTA, a pair-wise sequence alignment was performedagainst all Abs in the MSTA using BLAST, and the most similar sequence(highest E value) was identified. The Ab of interest was then structurallyaligned to this most closely sequence-related structure using CE, allowingthe mapping of its residues to the MSTA.

Finally, the RMSD and the changes in solvent accessibility and interchaincontacts were averaged, first over all pairs of either free–bound or bound–bound structures of the same Ab (to avoid bias toward Abs that have morestructures than others) and then over all Abs.

Space groups

The space group in which each structure was solved was extracted from“REMARK 290” in the PDB file. The position-based comparisons de-scribed above were then repeated, including only pairs of structures thatwere solved with the same space group.

Segment-based comparison

The CDRs were defined as described by Ofran et al. (48). The first loop ofthe constant heavy-1 (CH1-1) domain was defined between positions 159and 171 in the MSTA of Ofran et al. (48). The RMSD was averaged firstover all pairs of either free–bound or bound–bound structures of the samecluster and then over all Abs. For each two segments, the statistical sig-nificance of their average RMSD difference was computed using the fol-lowing reshuffling procedure: all RMSD values (one from each Ab) of thetwo segments were randomly divided into two new lists (of the same size asthe original ones), and the random average difference was calculated bysubtracting the average of one list from that of the other. This process wasrepeated 10,000 times, and the p value was calculated. The RMSD averagesof two segments for which p # 0.05 were considered to be significantlydifferent.

Heavy–light relative orientation

To compare the relative orientation of the H chain versus the L chain, weused a strategy similar to that of other investigators (4, 33, 38); we super-imposed two equivalent chains of the structures being compared andcalculated the Ca RMSD between the two other equivalent chains. Intra-domain changes are ignored by reducing the intradomain RMSD. Specifi-cally, for each pair of free–bound or bound–bound structures of the sameAb, the RMSDvariable and RMSDconstant, which estimate the change in theheavy–light relative orientation of the variable or of the constant domain,respectively, were calculated as follows (steps 1 and 2 are also illustrated inFig. 4):

1) The heavy variable domains of the two structures were structurallyaligned using CE, and the Ca RMSD between the two variable lightdomains (RMSDall) was calculated.

2) The light variable domains of the two structures were structurallyaligned using CE, and the Ca RMSD between these two variablelight domains (RMSDintra) was calculated.

3) RMSDvariablelight was calculated by subtracting RMSDintra from RMSDall.

4) Similarly, RMSDvariableheavy was calculated by applying steps 1–3 to

the other chain.5) The RMSDvariable is the average of RMSDvariable

light and RMSDvariableheavy .

RMSDconstant was calculated by applying steps 1–5 to the constantdomains instead of the variable domains. The RMSDvariable and theRMSDconstant were averaged first over all pairs of either free–bound orbound–bound structures of the same Ab, and then over all Abs. The pvalues were calculated as described above.

Variable–constant relative orientation

For each pair of free–bound or bound–bound structures of the same Ab,RMSDheavy and RMSDlight, which estimate the change in the variable–constant relative orientation of the H chain and the L chain, respectively, aswell as the corresponding p values, were calculated in a similar way to theheavy–light relative orientation described above.

Disordered residues

A residue was considered disordered if its coordinates were missing fromthe PDB file. The CH1-1 loop of a structure was considered disordered if atleast one of its residues is disordered.

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B-factor

The Ca B-factors of all residues were extracted from the PDB files. Theaveraged B-factor of the CH1-1 loop was calculated by averaging the B-factors over the loop positions (159–171), over all structures of the sameAb, and then over all Abs. For comparison, the B-factor was averaged overall other Fab positions (excluding the CH1-1 loop), as well.

Multiple sequence alignment of the CH1 domain

The sequence of all alleles of human IgG genes were downloaded from theInternational Immunogenetics Information System Web site (49) andaligned using ClustalW (50). The conservation scores for each positionwere calculated using JALVIEW (51, 52).

ResultsDataset

We retrieved all Abs with experimentally determined three-di-mensional structures in both free and bound forms. We consideredonly pairs of structures that share 100% sequence identity in both theH and L chains. Table I lists 141 PDB structures representing 49different Abs that make up our dataset. Thirteen of the 49 Abs hadmore than one bound structure, allowing the comparison of twobound structures. This bound–bound comparison provides a con-trol set that allows us to determine whether an observed change isAg-binding related: a conformational change may be consideredas binding related only if it is observed between free and boundstructures but not between different bound structures of the sameAb. Overall, our dataset consists of 94 free–bound and 162 bound–bound pairs of structures of the same Ab. Twenty-two of the Abswere bound to peptide Ags (#33 aa), and the other 27 were boundto proteins (the shortest of which was 104 aa). The sequenceidentity of the Ags bound to the same Ab in different structuresvaries in peptide Ags from 100% (e.g., 1hin versus 1him) to 0%(e.g., 1cfs versus 1hh9). In protein Ags, the sequence identityvaries from 100% (e.g., 1yyl versus 1yym) to 83% in the entire Agbut 95% in the epitope (e.g., 1rzk versus 2ny5). For 6 of the 49 Abs,at least one structure consists of only the variable domain, whichprecludes their use in calculating conformational changes in the con-stant domains and variable–constant relative orientation changes.

Position-based comparison

To identify the structural changes that occur at the residue levelbetween free and bound structures of the same Ab, we calculatedthe RMSD (once for Ca only and once for all heavy atoms), thechange in solvent accessibility, and the change in the number ofinterchain contacts for each position in each of these pairs ofstructures. The results from structurally equivalent positions of dif-ferent free–bound pairs of the same Ab and of different Abs wereaveraged. When possible, the same calculation was performed forpairs of bound structures of the same Ab to characterize thestructural changes as related to Ag binding or not. Fig. 1 shows theobserved changes, at the residue level, for free–bound (blue) andbound–bound (red) comparisons for the following features: CaRMSD (Fig. 1A), heavy-atoms RMSD (Fig. 1B), change in solventaccessibility (Fig. 1C), and change in the number of contacts withthe other chain (Fig. 1D). In this representation, two regions clearlystand out: CDR-H3 and the first loop of the CH1 domain (CH1-1loop) that is located between positions 159 and 171 and shown inFig. 2. For all features considered, the differences between free andbound in these regions were substantial. The conformationalchange in CDR-H3 appears only when comparing free and boundstructures and not when comparing two bound structures, sug-gesting that the conformational change is directly related to Agbinding. Changes in the CH1-1 loop are observed in both free–bound and bound–bound comparisons, suggesting, that in this case,at least part of the change may be ascribed to factors other than Ag

Table I. The dataset

ClusterNumber PDB Code H Chain L Chain Free/Bound

AgChainsa

ConstantDomainb

1 1vfa B A Free – 21 1vfb B A Bound C 22 1fgn H L Free – +2 1ahw B A Bound C +3 1mqk H L Free – 23 1ar1 C D Bound AB 23 1qle H L Bound ABCD 23 3ehb C D Bound AB 23 3hb3 C D Bound AB 24 1ay1 H L Free – +4 1bgx H L Bound T +5 1bey H L Free – +5 1ce1 H L Bound P +6 1bvl A B Free – 26 1bvk B A Bound C 27 1cfq B A Free – +7 1cfn B A Bound C +7 1cfs B A Bound C +7 1cft B A Bound C +7 1hh6 B A Bound C +7 1hh9 B A Bound C +7 1hi6 B A Bound C +8 1ck0 H L Free – +8 2ck0 H L Bound P +8 3ck0 H L Bound P +9 1cr9 H L Free – +9 1cu4 H L Bound P +10 1dqm H L Free – +10 1dqq B A Free – +10 1dqj B A Bound C +10 1nby B A Bound C +10 1nbz B A Bound C +11 1e6o H L Free – +11 1e6j H L Bound P +12 1f8t H L Free – +12 1f90 H L Bound E +13 1fvc B A Free – 213 1n8z B A Bound C +14 1gig H L Free – +14 2vir B A Bound C +14 2vis B A Bound C +14 2vit B A Bound C +15 1hil B A Free – +15 1him L H Bound P +15 1hin H L Bound P +15 1ifh H L Bound P +16 1kcu H L Free – +16 1kc5 H L Bound P +17 1kcv H L Free – +17 1kcs H L Bound P +18 1mf2 H L Free – +18 2hrp H L Bound P +19 1nlb H L Free – +19 1n64 H L Bound P +20 1mim H L Free – +20 3iu3 A B Bound K +21 1mlb B A Free – +21 1mlc B A Bound E +22 3d69 B A Free – +22 1nl0 H L Bound G +23 1oaq H L Free – 223 1ocw H L Free – 223 1oaz H L Bound A 224 1om3 H L Free – +24 2oqj B A Bound C +25 1qbl H L Free – +25 1wej H L Bound F +26 1rhh B A Free – +26 2b4c H L Bound CG +27 1rz8 B A Free – +27 1rzj H L Bound CG +27 1rzk H L Bound CG +

(Table continues)

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binding. The five other CDRs (CDR-H1, CDR-H2, and CDR-L1–L3) do not exhibit significant changes in any of the parametersrelative to the frameworks (FRs) or the constant domains. Theoverall similarity in the patterns of the Ca and heavy-atoms RMSD(Fig. 1A versus Fig. 1B) indicates that no specific region (includingthe CDRs) is characterized by movements of the side chains alone.It also indicates that free–bound changes are not enriched in back-bone or side chain rearrangements compared with bound–boundchanges, and vice versa.In the variable domain, six loops point toward the constant domain,

opposite the CDRs (three on each chain). Two of them, the second oneach chain (located at FR-2), show larger changes compared with theother four (Fig. 1A–D: residues 51–55 and 55–59 in the H and Lchains, respectively, and Fig. 1E: VH-2 and VL-2 loops). These twoloops interact with each other, and together with CDR-H3 and CDR-L3 they constitute a major part of the heavy–light interface (32);thus, their conformational changes may affect the heavy–light rel-ative orientation and, as a result, the Ag binding site itself.Overall, some periodicity can be observed in Fig. 1, including

some peaks that do not correspond to the CDRs or to the loopsdiscussed above. This periodicity is a result of the secondary struc-ture of the Ab scaffold, with low-flexibility regions correspondingto b-strands and peaks corresponding to loops. For all CDRs otherthan CDR-H3, the flexibility, as reflected by the peaks, is similarto that seen in nonbinding loops.In almost all positions, the RMSD between free and bound

structures is greater than the RMSD between two bound structures.A possible explanation we explored is that most free–bound pairs(74%) were solved in different space groups, whereas most of thebound–bound pairs (58%) were solved in identical space groups(Supplemental Fig. 1A). Thus, one might expect that free–boundpairs will more often have different crystal contacts, which may betranslated into structural diversity. To test this hypothesis, wereanalyzed the Ca RMSD only for pairs of free–bound and bound–bound structures solved in the same space group. However, theresults of this analysis indicate that the overall trend remains, withfree–bound changes clearly greater than bound–bound ones, in nearlyall Fab positions (Supplemental Fig. 1B). Thus, these differencescould not be ascribed solely to differences in space groups.

Segment-based comparison

To quantify the conformational changes that occur in specific seg-ments of interest (i.e., the CDRs and the CH1-1 loop) relative to eachother and to the rest of the Fab, we recalculated the Ca RMSD offree–bound and bound–bound pairs for each segment (instead of foreach position) separately. We also calculated the Ca RMSD of a“baseline” (i.e., all residues of the Fab chains, both variable andconstant domains) excluding the CDRs and the CH1-1 loop. Asshown in Fig. 3A, all CDRs exhibited greater changes in free–bound comparisons than in bound–bound comparisons (p , 0.001,p , 0.001, p , 0.001, p # 0.003, p # 0.008, and p # 0.002, forCDR-H1, -H2, -H3, -L1, -L2, and -L3, respectively). As suggestedin the position-based analysis, the baselines of both H and L chainsdiffer more when comparing free with bound than when comparingtwo bound structures (p , 0.001 and p # 0.001 for the baseline ofthe H and L chain, respectively). The free–bound average RMSDsof the CDRs and the baselines are still significantly higher than arethe bound–bound ones when including only pairs of structuressolved with the same space group (p # 0.005 for the CDRs, p #

0.024 for the baselines).The CH1-1 loop is the region in the Fab structure that exhibits the

largest conformational change, with an average Ca RMSD of 1.8 A(Fig. 3A). This value is significantly higher even than that of

Table I. (Continued )

ClusterNumber PDB Code H Chain L Chain Free/Bound

AgChainsa

ConstantDomainb

27 1yyl H L Bound MG +27 1yym H L Bound MG +27 2i5y H L Bound MG +27 2i60 H L Bound MG +27 2nxy D C Bound AB +27 2nxz D C Bound AB +27 2ny0 D C Bound AB +27 2ny1 D C Bound AB +27 2ny2 D C Bound AB +27 2ny3 D C Bound AB +27 2ny4 D C Bound AB +27 2ny5 H L Bound CG +27 2ny6 D C Bound AB +28 1u6a H L Free – +28 3hi1 H L Bound G +29 2zkh H L Free – +29 1v7m H L Bound V +29 1v7n H L Bound V +30 1yy8 B A Free – +30 1yy9 D C Bound A +31 2g75 A B Free – +31 2dd8 H L Bound S +32 2eh7 H L Free – +32 2eh8 H L Bound P +33 2fat H L Free – +33 2fd6 H L Bound AU +33 3bt2 H L Bound ABU +34 2fjf H L Free – +34 2fjg H L Bound WV +35 2pr4 H L Free – +35 3d0l B A Bound C +35 3d0v B A Bound C +35 3idg B A Bound C +35 3idi B A Bound C +35 3idj B A Bound C +35 3idm B A Bound C +35 3idn B A Bound C +36 2vxu H L Free – +36 2vxt H L Bound I +37 3cvi H L Free – +37 3cvh H L Bound ABC +38 3eo9 H L Free – +38 3eoa H L Bound I +38 3eob H L Bound I +39 3eyo B A Free – +39 3eyf B A Bound E +40 3gje B A Free – +40 3gjf H L Bound ABC +41 3hi5 H L Free – +41 3hi6 X Y Bound B +42 3pp3 H L Free – +42 3pp4 H L Bound P +43 1bbd H L Free – +43 1a3r H L Bound P +44 1igf J M Free – +44 2igf H L Bound P +45 1l7i H L Free – +45 1s78 D C Bound A +46 1mnu H L Free – +46 1mpa H L Bound P +46 2mpa H L Bound P +47 2gsg B A Free – 247 2otu B A Bound P 247 2otw B A Bound E 248 2hkh H L Free – +48 2hkf H L Bound P +49 3bkc H L Free – +49 3bkm H L Free – +49 3bkj H L Bound A +

aStructures that do not contain Ag chains are labeled “2”.bStructures that contain or do not contain the constant domain are labeled “+” or

“2,” respectively.

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CDR-H3 (p # 0.038). In addition, a change .1A in this loop iscommon for 63% of the Abs (Supplemental Fig. 2). The averageRMSD of free–bound is higher than that of bound–bound. As in-dicated by the error bars in Fig. 3A, the variability of the RMSDsin this loop in different Abs is high. The difference between free–bound and bound–bound has a p value # 0.045. However, whenincluding only pairs of structures solved with the same spacegroup, the p value increases to #0.357. Thus, it is impossible toestablish that the changes of this loop are related to Ag binding.The flexibility of CH-1 is also reflected by the observation that itsbound–bound average RMSD is significantly higher than that of

the other CDRs and the baseline (p # 0.002). We also found that37% of the structures in our dataset have intrinsic disorder in thisregion (Supplemental Table IA) and that the averaged B-factor ofthis loop is high compared with the rest of the Fab (SupplementalTable IB).Of the CDRs, CDR-H3 has the highest free–bound average

RMSD (1.30 A), which is significantly greater than the baseline(p, 0.001). Nonetheless, the free–bound RMSD of CDR-H3 is.1A in only 37% of the Abs (Supplemental Fig. 2) (i.e., a largeconformational change of this CDR upon Ag binding is not acommon feature of most Abs). However, when such changes in

FIGURE 1. Position-based com-

parison of free–bound and bound–

bound structures. Ca RMSD (A),

heavy-atoms RMSD (B), change

in solvent accessibility (C), and

change in contacts with the other

chain (D). The H chain residues are

presented in the left panels, and the L

chain residues are presented in the

right panels. Free–bound compar-

isons are shown in blue, and bound–

bound comparisons are shown in red.

The CDRs are presented as plus signs,

and all other residues are depicted as

dots. Positions for which the data

originated from fewer than three

Abs are not shown, as well as the el-

bow region connecting the variable

and constant domains and the struc-

ture edges. (E) The structure of BH-

151 Ab (PDB ID: 1eo8) is colored

according to the average free-bound

Ca RMSD values shown in (A), from

blue (low RMSD values) to red (high

RMSD values). Regions for which

the RMSD was not calculated are

colored black. b-strands and loops

are presented as wide and narrow

ribbons, respectively.

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the conformation of CDR-H3 were observed, they occurred be-tween the free form and any of the bound structures (as opposedto between the free structure and only some of the bound ones).In contrast to the free–bound change, the bound–bound changein CDR-H3 is not significantly higher than that of the other CDRsand the baseline (p # 0.312), suggesting that when conformationalchanges occur in CDR-H3 they are related to Ag binding.

The free–bound average RMSDs of CDR-H1, -H2, -L1, and -L3are ∼0.5 A and are not significantly different from those of thebaselines (p # 0.47, p # 0.41, p # 0.27, and p # 0.24, respec-tively). Nevertheless, there are some specific examples in whichthe RMSD for these CDRs is high. Specifically, the RMSD of CDR-H1, CDR-H2, CDR-L1, and CDR-L3 was .1 A in 12, 14, 4, and4% of the Abs, respectively (Supplemental Fig. 2). The free–bound

FIGURE 2. The CH1-1 loop in the Ab scaf-

fold. (A) HyHEL-63 Ab CH1 and CL constant

domains (PDB ID: 1dqm). The CH1 and CL

domains are colored cyan and light green, re-

spectively. The CH1-1 loop residues and the CL

residues within 5 A of the CH1-1 loop are

presented as sticks. The Cys of the CH1-1 loop

and the Cys of the CL domain connected by an

S-S bond are colored by element. (B) The entire

Ab scaffold (PDB ID: 1igy). The H and L

chains are colored blue and orange, respec-

tively. The CH1-1 loop is presented as a space-

filling representation and is colored light green.

FIGURE 3. Intra- and interdomain changes. (A) Segment-based comparisons. Dark and light gray bars represent the Ca RMSD of free–bound and

bound–bound pairs, respectively. Standard errors are shown as error bars. (B) Average heavy–light relative orientation change, in the variable and constant

domains, of free–bound (dark gray bars) and bound–bound (light gray bars) pairs. (C) Average variable–constant relative orientation change, in the H and L

chains, of free–bound (dark gray bars) and bound–bound (light gray bars) pairs. Standard errors are shown as error bars.

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average RMSD of CDR-L2, 0.35 A (Fig. 3A), is significantly lowerthan that of all other CDRs and even than the free–bound RMSD ofthe baseline (p , 0.001).

Heavy–light relative orientation

Next, we characterized free–bound and bound–bound changes inthe relative orientation of the H and L chains (see Materials andMethods and Fig. 4). Fig. 3B shows that the change in the heavy–light relative orientation of both variable and constant domains issignificantly higher in free–bound comparisons than in bound–bound comparisons (p , 0.001 and p # 0.001 for the variable andconstant domains, respectively). This observation suggests that Agbinding affects the relative orientation of the H and L chains bothin the variable domain and in the constant domain. The change inthe VH–VL relative orientation is significantly higher than that ofCH1-constant light (CL) when comparing free and bound struc-tures (p , 0.001) but not when comparing two bound structures(p # 0.07). Thus, we conclude that the higher conformationalchange in VH–VL (relative to that of CH1-CL) is the result ofAg binding. However, this effect is relatively minor, even in thevariable domain, with an average RMSD of 0.58 A and a maximumvalue of 1.76 A.

Variable–constant relative orientation

In a similar way, we also characterized the relative orientation ofthe variable versus the constant domain. Fig. 2C shows that thevariable–constant relative orientation change in free–bound com-parisons is remarkable (average RMSD of 3.51 and 3.60 A, and amaximum value of 17.88 and 19.01 A for the H and L chain, re-spectively), compared with the change observed in heavy–lightrelative orientation. It seems that the change in variable–constantrelative orientation is a result of the Ag binding, because the av-erage RMSD of the free–bound comparisons is significantly higherthan that of the bound–bound comparisons (p , 0.001 for both Hand L chains). Variable–constant relative orientation change issimilar in the H and L chains.Because the Ag binding may induce such a significant change in

the variable–constant relative orientation, we hypothesized thatthis effect may depend on the Ag size. Fig. 5 shows that, indeed, theaverage RMSD of variable–constant relative orientation inducedby protein Ags is significantly higher than the change induced bypeptide Ags (p # 0.002). Although the type of Ag cannot be triv-ially used for predicting the amount of change in the variable–

constant relative orientation, because some of the protein Ags stillshow a very small change (e.g., 0.30 A between 3gje and 3gjf),dramatic changes can almost be excluded for peptide Ags, because18 of these 21 Ags do not induce a change that is .1.74 A. Surpris-ingly, a similar relationship between the Ag size and the heavy–light relative orientation was not found, nor were such relation-ships found between the Ag size and the CDR-H3 RMSD or betweenthe Ag size and the CH1-1 loop RMSD.

DiscussionIn 1993, when the first comparative studies of Abs appeared (32, 34),the structures of only 33 Abs were available. With the structures ofalmost 1400 Abs available in the PDB, we attempted to reassessbinding-related conformational changes. Of the six CDRs, CDR-H3 shows the largest conformational change upon binding the Ag,as was previously suggested (30, 32, 36). It has the highest numberof contacts with the Ag (53) and is believed to play a key role inAg recognition (54). It is also the only CDR that shows a consid-erable conformational change ($0.6A Ca RMSD) when compar-ing free and bound germline Abs (39). It was suggested (39) thatthis flexibility of CDR-H3 in germline Abs enables the binding ofdifferent Ags, and that during affinity maturation, it becomes morerigid with a conformation that is optimal for a specific Ag. Ourresults indicate that in many of the Abs, CDR-H3 shows only minorconformational changes between free and bound forms. However,despite the rigidification of the Ab during affinity maturation, CDR-H3 still shows substantial structural changes in 37% of the Abs andan average Ca RMSD of 1.3 A between free and bound structures(and sometimes this RMSD is .3 A). None of the other CDRsdisplayed a conformational change greater than the changes ob-served for the rest of the Fab structure (the FRs and the constantdomains). CDR-L2 shows the smallest structural change upon Agbinding. Notably, this change is significantly lower than that of theother CDRs and is even smaller than the change in the FR and theconstant domain (the baseline). It is not the shortest CDR, but it hasthe lowest number of contacts with the Ag (53), a low number ofmutations during affinity maturation (55), and low structural di-versity in different Abs (56). These observations may suggest thatthe conformational change that a CDR undergoes is related to itsrole in Ag binding.Structural changes upon Ag binding also occur in the relative

orientation of the Fab domains. Such interdomain movements weresuggested to affect protein functions in other systems as well (38).

FIGURE 4. Schematic illustration of the first two

steps in calculating the change in VH–VL relative

orientation (see Materials and Methods). (A) The two

structures are superimposed according to the variable

domain of the H chains, and RMSDall is calculated

based on the L chain variable domains. (B) The two

structures are superimposed according to the variable

domain of the L chains, and RMSDintra is calculated

based on the L chain variable domains. The PDB IDs

of the two structures are 1e6o (green–orange) and 1e6j

(blue–red). The H chains are colored green and red,

and the L chains are colored orange and blue.

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More than 20 y ago, Colman et al. (57, 58) suggested that the Agbinding site may be shaped by repositioning of the VH domainwith respect to the VL. This suggestion, further supported by morerecent studies (4, 29, 30, 38), highlights the significance of un-derstanding the relative orientation of these two chains. We foundthat the relative orientation of the H–L chains changes more whencomparing free–bound structures than when comparing bound–bound structures, in both the variable and the constant domains.These findings are in agreement with the idea that the VH–VLrelative orientation contributes to Ag binding, but they also suggestthat the CH1-CL relative orientation shows a similar (yet lessprominent) effect. The observation that the loops in the heavy andlight FR-2s, which play an important role in the heavy–light in-teraction (32), show some conformational change, further dem-onstrates the relationship between the VH–VL interface and Agbinding. With this in mind, the actual change in free–bound relativeorientation of VH versus VL is still limited, in agreement with asmall rotation ,3˚ observed previously (33). The Ag-binding–re-lated changes observed in VH–VL relative orientation are smallerthan the difference in the related orientation observed betweendifferent Abs (38).The relative orientation of the variable–constant domains in both

the H and the L chains undergoes a more prominent change thandoes the VH–VL domains. As in the case of heavy–light, the changeobserved in free–bound is greater than the change observed inbound–bound, indicating that these changes are related to Agbinding. Changes in the elbow angle as a result of Ag binding werealso suggested by a molecular dynamics simulation (59). The largechange in the variable–constant relative orientation does not nec-essarily support the already disproved theory of an “open” confor-mation for a free Ab and a “closed” one for the bound form (30);rather, it demonstrates the high flexibility of the Ab, introduced bythe elbow region between the variable and the constant domains.This flexibility, which results in a more significant conformationalchange in the elbow angle than in the VH–VL relative orientationupon Ag binding, was also suggested to occur when comparingdifferent Abs (as opposed to different structures of the same Ab)(32). What could be the role of this flexibility? A change in thevariable–constant relative orientation may change the contacts be-tween residues in these two domains (32), and this may change theVH–VL relative orientation, shaping the binding site for Ag bind-ing. Alternatively, Ag binding may cause a change in the VH–VLrelative orientation, which, in turn, may change the contacts be-tween residues in the variable–constant interface, resulting in adifferent elbow angle. This different elbow angle could be translated

into a different CH1-CL relative orientation and possibly enable ordisable the binding of an effector to the constant domains. Thesemechanisms suggest that a relationship between the change inelbow angle and Ag binding is plausible, as observed in our results.Additional support for this relationship is the dependence of theelbow angle change on the Ag size: when the Ag is a protein, theaverage change in the elbow angle between free and bound structuresis more than three times the change observed for peptide Ags.Surprisingly, the region showing the greatest and most consistent

conformational change between free and bound structures is theCH1-1 loop, which is located in the constant domain, far from theAg binding site. Although we were not able to prove that the con-formational changes in the CH1-1 loop are related to Ag binding,because substantial conformational changes were observed in thisloop in bound–bound comparisons as well, some of its character-istics, as well as several previous results, suggest that it may havea role in Ab function. As shown in Fig. 2A, this loop is part of theinterface with the CL domain; in some cases, it is even connected tothe CL domain through a disulfide (S-S) bond, thereby having apotential effect on the CH1-CL relative orientation. Furthermore,this loop is close in space to the hinge region connecting the CH1domain to the Fc (Fig. 2B). Thus, a conformational change in thisloop may affect the relative orientation of the CH1 domain versusthe Fc, thereby influencing effector binding to the Fc. There are nowmany examples of Abs with identical variable domains but differentisotypes that bind the same Ag with a different affinity or specificity(13, 15–24). Some of these studies suggested that the CH1 domainaccounts for the observed changes in binding, because it wasidentified as the only region with sequence diversity between thetested Abs and because a similar effect was observed using onlythe Fab instead of the entire Ab scaffold (13, 16, 19). SupplementalFig. 3 shows a multiple sequence alignment of all human IgG CH1alleles. Sequence diversity within this domain is observed in onlytwo regions, one of which is the CH1-1 loop, suggesting that it mayplay a role in the functional diversity of Abs. In addition to itsflexibility, the CH1-1 loop is also intrinsically disordered, at leastin part, in more than one third of the structures in our dataset,which may suggest the existence of a functional binding site (60).Although the majority of Ab effector functions (e.g., complementactivation and interaction with FcRs) are mediated by binding ofthe Fc, it was suggested that complement binding is also mediatedby the CH1-1 loop. In particular, C3b was shown to covalentlybind Ser132 [Eu numbering (61)], which belongs to the CH1-1 loop,during complement binding to the Ab–Ag complex (62). Anotherexample of the potential role of the CH1 domain in Ab activity isthe lack of ability to activate the alternative pathway of comple-ment by IgG molecules with the inter–CH1-CL S-S bond reduced(63). A possible mechanism for the effect of a conformationalchange in the CH1-1 loop on Ag binding could be a change in theCH1-1 loop microenvironment, as a result of effector binding ora change in the hinge region, which may result in a change to theCH1-CL relative orientation. As discussed above and suggestedpreviously (26, 28, 62), this may lead to changes in the VH–VLrelative orientation through the elbow angle and, thus, to changesin the Ag binding site. Such a mechanism is also consistent witha recent analysis that found a large difference in affinity betweena Fab and its corresponding Fv fragment (12). Similarly, a signalmay be transferred following Ag binding, from the variable, throughthe variable–constant interface and the elbow angle, to the CH1domain, and possibly through the hinge region to the Fc domains.Additional studies may shed more light on the nature of this loopand its role, or lack thereof, in the immune response.It is generally accepted that Abs do not bind Ags in a rigid lock-

and-key manner but rather exist in multiple conformational states

FIGURE 5. The variable–constant relative orientation change in peptide

versus protein Ags. Standard error bars are shown.

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(8). However, it is unclear whether the Ag binding induces thechange or binds to pre-existing conformation [“pre-existing equi-librium” (3, 35)]. Although structural changes between free andbound Abs were commonly associated with an induced-fit mech-anism, structural evidence for the pre-existing equilibrium wasfound for SPE7 Ab (35); two pre-existing conformations of the Ag-free form were crystallized, each conferring a different Ag-bindingfunction. A normal-mode analysis study of two Abs (3), showingthat the intrinsic fluctuations of the free conformation correlatewith the structural changes observed upon Ag binding, is also inagreement with this hypothesis. Our finding, that some Abs showa significant conformational change upon Ag binding whereasothers do not, can be explained by the existence of multiple freeconformations in equilibrium, some of which are capable of bindingspecific Ags. If the free conformation that was crystallized is thesame one bound to the complexed Ag, only minor structural changesbetween the free and bound structures will occur. However, if adifferent pre-existing free conformation was crystallized, signifi-cant changes are expected. Additional analyses of Abs with morethan one free structure, as well as bound ones, will further revealthe extent to which this hypothesis is supported.It is believed that a single Ab may bind multiple Ags, because

Abs have a limited repertoire of structures yet can theoreticallybind an infinite number of Ags (3). It was suggested that this abilityis facilitated by the Ab conformational flexibility (3). Thus, onemay expect the similarity of two Ags, and the structural changesthat occur in the Ab that binds them both, to be correlated to someextent. Multiple bound structures of the same Ab, with somevariability in the Ag epitopes, are required to search for such acorrelation. Our dataset includes only two such clusters. Indeed, inone of these clusters, a correlation was observed between epitopesequence similarity and the resulting changes in the Ab struc-ture (data not shown). New structures solved in the coming yearsare likely to provide additional examples of multiple structures ofdifferent Ags bound to the same Ab and will enable a morecomprehensive analysis in this regard.A potential concern when comparing free and bound crystal

structures is the existence of possible experimental factors thataffect free or bound structures in different ways, which may lead tosome artifacts. For example, Fabs tend to crystallize in a similarlattice. Crystallization of Fab–Ag complexes (especially when theAg is large) is likely to occur in a different lattice. We attempted toaddress this concern by analyzing only pairs of structures that weresolved with the same space group. Notably, comparing onlystructures that have similar unit cell sizes may have been moreaccurate; however, there are no complexes of Abs bound to a pro-tein Ag that have a similar unit cell size as the free Ab. An addi-tional potential artifact may result from the fact that, in general,Ab–Ag complexes tend to have more biological impact than dotheir free counterparts. This may cause crystallographers to investmore effort into trying to solve bound structures, which may resultin the use of unusual crystallization conditions. A possible way toavoid such an artifact is the comparison of only pairs of structuresthat were both solved in common space groups, as suggestedpreviously (64). However, the current dataset does not allow forsuch analyses because there are hardly any pairs of this sort. Thus,we presented our analysis, keeping in mind these potential biases.mAbs represent a growing segment of biological drugs for

treating a variety of diseases (5). Ab structures, and specifically Ab–Ag complexes, can help to overcome challenges in Ab stabiliza-tion, affinity maturation, and humanization, all of which are re-quired for the successful development of therapeutic Abs (7).When a structure of the Ab–Ag complex does not exist, it can bemodeled with computational docking tools, relying on the struc-

tures of the monomers. However, for an accurate Ab–Ag docking,the structural changes occurring in the Ab and Ag upon bindingshould be recognized. Indeed, the inherent flexibility of proteins isconsidered one of the most challenging topics in molecular model-ing (9). The comprehensive characterization of free–bound changesin Abs provided in this article may to help improve Ab modelingand Ab–Ag docking algorithms by incorporating the conclusionspresented in this study into the modeling techniques and dockingalgorithms.

AcknowledgmentsWe thank Turkan Haliloglu, Sharon Fishman, Anat Burkovitz, Guy Nimrod,

Vered Kunik, and Ariel Feiglin for valuable comments and suggestions.

DisclosuresThe authors have no financial conflicts of interest.

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10 BINDING-RELATED CONFORMATIONAL CHANGES IN Abs

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