Conserved sequence and structure association motifs in antibody–protein and antibody–hapten complexes

Dennis R. Livesay1,2 and Shankar Subramaniam3,4

1Department of Chemistry and National Center for Supercomputing Applications, University of Illinois at Urbana–Champaign, Urbana, IL 61801 and 3Department of Bioengineering, Department of Chemistry and Biochemistry and San Diego Supercomputing Center, University of California at San Diego, La Jolla, CA 92037, USA

4 To whom correspondence should be addressed. E-mail: shankar{at}ucsd.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this paper, we present the association requirements across a wide variety of antibody–antigen complexes. Phylogenetic analysis clearly indicates the representative nature of our structural dataset. Antigen molecules range from small-molecule haptens to complete protein structures. Common association motifs identified include five conserved tyrosine residues and a single conserved arginine residue from CDR-H3. Further, specificity is refined by a diverse array of antibody–antigen electrostatic interactions that maximize complex specificity. Through analysis of calculated pKa shifts on antigen binding, we find that these interactions are conserved at 23 alignment ‘hot-spot’ positions. Despite consistent roles in defining substrate specificity, 16 hot-spot positions are conserved less than 50% of the time. On the other hand, because of the conserved functional role of these positions, mutant screening at hot-spots is more likely to result in increased antigen specificity than elsewhere. Therefore, we believe these results should facilitate subsequent antibody design experimentation.

Keywords: antibody–antigen complex/antibody–hapten complex/immunoglobulin superfamily/molecular recognition


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The overall topology of the antibody (Ab) molecule is highly conserved. Each Ab molecule is composed of four [two light (L) and two heavy (H)] protein chains and each chain is composed of repeats of a distinct structural domain. The distinct immunoglobulin (Ig) domain is characterized as a seven-stranded Greek-key ß-barrel. Three-dimensional structures for several Ab Fab functional units and Ab–antigen complexes have been solved (for a review, see Padlan, 1994Go). The Fab fragment is composed of four Ig domains (two each from the light and heavy chains). The Fab fragment can be further reduced to the Fv fragment, which is the antigen recognition domain. The Fv fragment is composed of two Ig domains (one each from the light and heavy chains). Antigen recognition is generally mediated by 15–20 amino acids (Poljak et al., 1973Go; Davies and Metzger, 1983Go; Padlan, 1994Go), most of which are from six peptide loops that make up the complementary determining region (CDR). The CDR binding site is formed by loops connecting the anti-parallel ß-strands of the light and heavy chain variable region (Chothia and Lesk, 1987Go). Across the Ig superfamily, CDR sequences are highly variable, whereas more conserved residues make up the core of the Ab structure.

Point mutants at combining site residues of the Ab or antigen generally result in large specificity losses (Knossow et al., 1984Go; Alegre et al., 1992Go; Chacko et al., 1995Go). Charged residues frequently mediate Ab–antigen association, hence pH, ionic strength and the presence of cosolutes affect association specificity. Continuum electrostatic theory has been used to study a wide variety of Ab–antigen and Ab–hapten complexes (for a review, see Gibas et al., 2000Go). The method provides detailed pKa data for each titratable residue, in addition to overall pH-dependent energetics. We have thoroughly examined two Ab–lysozyme complexes (Slagle et al., 1994Go; Gibas et al., 1997Go) and one Ab–hapten complex (Livesay et al., 1999Go) using such methods. In all three examples, association is mediated by complementary charge–charge interactions at the combining site. Therefore, the local environment surrounding the combining site has a significant effect on the association energetics. Factors affecting association energetics include minor structural rearrangements, buried interfacial area, dielectric environment of key residues and geometry of the interacting residues. Comparisons of calculated pH-dependent behavior show good agreement with experimental results.

In addition to providing an overall titration profile, continuum methods quantitatively assess the relative influence of individual charged groups. Models of site-directed mutants have been constructed to probe the influence of each charged interface residue (Slagle et al., 1994Go; Gibas et al., 1997Go; Livesay et al., 1999Go). Examination of the electrostatic contribution to the association energy in mutant complexes confirms that charge complementarity at the combining site is an important requirement for antigen binding. Also, aromatic {pi}-stacking and van der Waals contacts between Ab and antigen residues contribute to specificity.

Experimental structures for a host of Ab–antigen complexes have now been solved, allowing the comprehensive investigation of a wide variety of Ab–antigen interfaces. Vargas-Madrazo et al. (1995)Go used the canonical structure model to conclude that the antibody structural repertoire is limited to a small number of canonical structural classes. Two sets of preferential canonical classes were identified, each associated with a particular antigen specificity. MacCallum et al. (1996)Go compared the topology of ten structural pairs of antigen-bound and apo Ab structures. They found that Ab binding sites could be divided into four topographical classes: concave, moderately concave, rigid and planar. Further, four of the 10 binding sites changed class on antibody binding. Several other studies have attempted to analyze antigen specificity vis-à-vis a variety of phenomena, including somatic hypermutation (Ramirez-Benitez and Almagro, 2001Go), CDR length and sequence composition (Collis et al., 2003Go; Almagro, 2004Go) and conformational changes on binding (Pellequer et al., 1999Go). Here, we used continuum electrostatic methods to probe thoroughly the electrostatic association requirements of a wide variety Ab–protein and Ab–hapten complexes. Residues that show significant changes in pKa values on complex formation are scrutinized to understand the chemical basis of each shift. Further, results from all complexes are compared in order to identify conserved association motifs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Calculation of protein pKa values

The continuum electrostatic methods used here are included in the University of Houston Brownian Dynamics (UHBD) suite of programs (Madura et al., 1995Go). For all calculations, partial charges were taken from the CHARMM parameter set (Brooks et al., 1983Go) and radii from the Optimized Parameters for Liquid Systems (Jorgensen and Tirado-Rives, 1988Go). Partial charges of non-protein substrates were calculated using Gaussian 96 with the 6–31G basis set. Model pKa values were taken from Antosiewicz et al. (1994)Go. Temperature, ionic strength and ionic radius remained constant throughout all calculations at 298 K, 150 mM and 2.0 Å, respectively. A solvent and interior dielectric of 80 and 20, respectively, (Antosiewicz et al., 1994Go; Gibas and Subramaniam, 1996Go) were used throughout. Finite grid spacing began at 1.5 Å and was focused to 1.2, 0.75 and 0.25 Å. Using a probe radius of 1.4 Å, the boundary between solvent and protein dielectrics was differentiated using the method of Shrake and Rupley (1957)Go.

Antibody structures

Ab structures used in the pKa calculations are modified versions of the coordinates available from the Protein Data Bank (Bernstein et al., 1977Go). Currently, there are 459 Ig structures stored within the Protein Data Bank with resolutions better than 3.0 Å; 348 of those are solved in the antigen-bound form. However, many of these structures are either redundant or theoretical models. A non-redundant dataset (eliminating mutant equivalent structures and theoretical models) reduces the number of structures to 97. In this work, our dataset is composed of 25 Ab–antigen complexes (13 Ab–protein and 12 Ab–hapten) taken from Ioerger et al. (1999)Go (Table I). Figure 1 shows phylogenetic trees that describe the diversity within the entire antibody structural proteome (antigen-bound or otherwise). Figure 1 also clearly indicates that our dataset is representative of that diversity and that general conclusions from our analysis can be made.


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Table I. Antibody Fv fragment–antigen complexes examined in this study

 


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Fig. 1. Phylogenetic trees of known antibody structural proteome. The distribution of protein structures analyzed here, indicated by hash marks to the right of the trees, clearly demonstrates the representative nature of our dataset.

 
Structures were modified as reported in the previous work (Gibas and Subramaniam, 1996Go; Livesay et al., 1999Go) to only include the Fv fragment and substrate. The single-site titration pKa calculation (Gilson, 1993Go; Antosiewicz et al., 1994Go) implemented in UHBD (Madura et al., 1995Go) used explicit polar hydrogen atoms. Polar hydrogens were added using the HBUILD program within Quanta96. Detailed analysis of the Ab–substrate interface region was done using LIGPLOT (Wallace et al., 1995Go).

Bioinformatics

Sequence alignment was based on structural superposition using the All-to-All Protein Structure Alignment server from the San Diego Supercomputer Center (Shindyalov and Bourne, 1998Go). Consensus sequence was determined using CLUSTALW (Thompson et al., 1994Go). Probability density functions (PDFs) counted the occurrence of target atoms within a 16 Å sphere about the mean hapten center of mass position. Residue target atoms were either the titrating atom (NZ of Lys; OH for Tyr) or atoms central to resonance-equivalent charged atoms (CG of Asp, CD of Glu, CZ of Arg and CD2 of His). CLUSTALW (Thompson et al., 1994Go) was used to align sequences from the complete antibody structural proteome. This alignment was used to compare the results from our representative dataset. Further, our results were compared with the Kabat database using KabatMan (Martin, 1996Go). The Kabat numbering scheme is used throughout.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antigen and hapten substrate structures

Substrate antigens investigated are divided into two classes, protein-based and small-molecule haptens. Protein antigens are further subdivided into complete structures and fragments. Four of the five complete protein antigens are lysozyme orthologs (all have 129 residues); the remaining one is a histidine-containing protein (85 residues). Protein fragments range in length from 7 to 12 residues and contain nearly identical contacts as the complete protein–antigen complexes. One Ab (1IKF) included here is specific for the cyclic peptide cyclosporin; we classify cyclosporin as a protein fragment. Table II summarizes the interfacial region of each Ab–antigen complex. In general, there are two less interfacial H-bonds and one less interfacial tyrosine residue in each of the Ab–protein fragment complexes. The number of salt bridges per complex (regardless of antigen type) is nearly constant.


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Table II. Analysis of antibody–protein combining site

 
Non-protein hapten molecules can also be subdivided into two subclasses. In the first, association is mainly mediated by aromatic {pi}-stacking interactions. The majority of these haptens are nitrophenyl derivatives (Figure 2). Many of the nitrophenyl derivatives also have polar functional groups involved in complex specificity. The remaining haptens are structurally and chemically variable and include steroids, a trinucleotide, a trisaccharide and various charged organic molecules. Table III summarizes the interfacial region in each Ab–hapten complex. Compared with Ab–protein complexes, there is much more variability in the number of H-bonds in Ab–hapten complexes. On the other hand, the number of salt bridges in Ab–hapten complexes is similar to those identified in Ab–antigen complexes. Discrimination between the two hapten subclasses occurs mainly in the number of tyrosine residues within the interfacial region. On average, there is one extra tyrosine residue at the combining site of antibodies specific to nitrophenyl compounds.



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Fig. 2. Schematic representation of combining site region for each antibody–hapten complex. Analysis of the antibody–hapten interface reveals similar numbers of interfacial tyrosine residues, hydrogen bonds and salt bridges.

 

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Table III. Analysis of antibody–hapten combining site

 
Changes in pKa values on association

Figure 3 maps calculated pKa shifts to sequence. The shifts are generally isolated to CDRs. In a previous study (Livesay et al., 1999Go), we found that there is a discrete number of pKa shifts in the monoclonal antibody (mAb) NC6.8. The shifts are localized to titrating residues at the combining site, whereas those further away remain unchanged. This result is generally conserved in all Ab–antigen complexes investigated here. Figure 4 shows the number of pKa shifts occurring at each position in the alignment. The pKa shifts are isolated to a handful of positions in much the same way as seen in the mAb NC6.8 study.




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Fig. 3. Structure-based sequence alignments of the (A) light and (B) heavy chains. The alignments are color-coded to indicate calculated acidic (gray) and basic (black) pKa shifts on association. Antibodies specific to protein antigens have bold titles, whereas those specific to hapten molecules do not. CDR regions are indicated above the alignment by Xs; consensus is indicated below the alignment.

 


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Fig. 4. Histogram tabulating the number of pKa shifts occurring at each position in the structural alignment of the (A) light and (B) heavy chains versus sequence consensus. Black indicates basic shifts and gray indicates acidic shifts. Twenty-three alignment positions have conserved pKa shifts (more than five). Tyrosine is the consensus residue for five of the 23 ‘hot-spots’. In CDR-H3 there are two ‘hot’ positions with consensus, one an arginine and the other an aspartate. The remaining ‘hot-spots’ have no consensus.

 
In mAb NC6.8, five residues are implicated as defining hapten specificity. Three of those residues (Glu H:35, Glu H:50 and Arg H:56) are involved in salt bridges with the guanidinium and acetic acid moieties of the hapten, whereas the other two residues (Tyr L:36 and Tyr L:96) are involved in {pi}-stacking and dipole–dipole interactions. Glu H:35 and Glu H:50 are also critical to antigen binding in the mAb HY/HEL10–lysozyme complex (Gibas and Subramaniam, 1997Go). Conservation in the association requirements between such dissimilar antigens is intriguing and was the initial motivation for this study. No other equivalents of Glu H:35, Glu H:50 or Arg H:56 occur in the other antibodies studied here. Three chemically conservative Arg to Lys mutations occuring at L:96 are observed; a significant pKa shift occurs on association in each. Tyr L:36 is highly conserved across our structural dataset (occurring 84% of the time). A significant pKa shift of Tyr L:36 occurs on association 64% of the time. Although not directly interacting with the hapten, a significant pKa shift also occurs at Tyr L:32 on formation of the mAb NC6.8–hapten complex. This residue and its pKa shift are highly conserved. In fact, the Tyr L:32 pKa shift is the second most conserved. Tyr L:32 occurs within CDR-L1 and is frequently hydrogen bonded to the substrate.

The remaining alignment positions undergoing changes in pKa on association are largely isolated to the CDRs. There are significantly more (67.8% more) pKa shifts occurring on the heavy than on the light chain. It follows that there are also more individual alignment positions with higher total pKa shift numbers. These ‘hot-spots’ are defined as alignment positions with total pKa shifts more than 1.5 standard deviations beyond the average. There are 15 so-called ‘hot-spots’ on the heavy chain, whereas there are only eight on the light chain. Tyrosine is the consensus residue for five of the 23 ‘hot-spot’ positions. There are two hot-spots in CDR-H3; one is a highly conserved arginine and the other is an aspartate residue. None of the remaining 16 hot-spots are conserved more than 50%, our criterion for consensus.

Probability density functions

Probability density functions (PDFs) describe the spatial distribution of a particular residue or atom about some target. At close distances (e.g. less than 5 Å), PDFs describe the contact probability between residue and target across our dataset, meaning that PDFs are an efficient way to collate visually multiple structural contact analyses. At larger distances (e.g. 10–15 Å), PDFs describe the likelihood of residues being spatially distributed around the substrate beyond normal contact cutoffs. Frequently, conserved features at larger distances describe spatially conserved residues one shell of residues removed from the target. We have used PDFs to probe the conserved spatial distribution of charge across multiple enzyme families and superfamilies (Livesay et al., 2003Go). In each of the cases investigated, the spatial distribution of charge is conserved within the active site. No conservation in the spatial distribution of charge or the spatial distribution of pKa shifts is observed across our Ab–antigen dataset. This result is surprising in view of the general importance of acid–base interactions at the Ab-combining site. However, analysis of the spatial distribution of tyrosine residues (specifically, tyrosine hydroxyl oxygens) at the combining site does yield interesting results. Figure 5 indicates that the overall shape of the tyrosine PDFs is conserved. This result advocates that tyrosine structural positions are more conserved than acidic and basic residues.



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Fig. 5. Stacked tyrosine hydroxyl probability density functions (PDFs) indicating all tyrosine residues occurring within the combining site of each structure. The PDFs are created by tabulating the occurrence of tyrosine hydroxyl oxygen atoms occurring within 16 Å of the mean hapten center of mass position. The shapes of the PDFs are qualitatively conserved across all structures.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein antigen versus hapten specificity

Comparison of the interfacial regions between anti-protein and anti-hapten antibodies reveals few novel results. Numbers of aromatic residues and salt bridges are nearly constant. The average number of hydrogen bonds does, however, vary between anti-hapten (2.4) and anti-protein antibodies (6.2). There is also a 33% increase in pKa shifts from anti-protein to anti-hapten antibodies. These differences are a reflection of the size differences (and therefore contact probability differences) between large protein antigens and small-molecule haptens. Changes in solvent exposure on protein binding also contribute to the increased pKa shift number. Otherwise, no consistent differences leading to Ab specificity between antigen classes are observed.

Combining site tyrosine residues

Complex formation significantly affects the pKa value of five conserved tyrosine residues. These residues are located at L:32, L:36, L:49, H:27 and H:32. The three light-chain tyrosines are highly conserved across our structural dataset (76, 76 and 84%, respectively). The two heavy-chain tyrosines are more variable and commonly substituted by phenylalanine. The tyrosine residue that occurs at L:36, just past CDR-L1, has the most conserved pKa shift of any alignment position. This is noteworthy for three reasons: (1) the residue occurs outside the CDRs; (2) the residue is buried deep in the binding pocket of the Fv fragment; and (3) the residue displays a large amount of conformational freedom (see Figure 6A). Despite the observed conformational variability, the crystallographic temperature factor of each residue is low. The role of the conserved Tyr L:36 appears to be mostly electrostatic. For example, in the mAb NC6.8–hapten complex, Tyr L:36 forms a stabilizing dipole–dipole interaction with the cyanophenyl moiety of the hapten. The spatial freedom maximizes the electrostatic interactions between the tyrosine residue and substrate (see Figure 6B).



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Fig. 6. (A) The highly conserved Tyr L:36 (mAb NC6.8 numbering) displays a large amount of conformational freedom, despite being buried deep in the binding pocket of the Fv fragment. In each structure the temperature factor of this residue is low. (B) Tyr L:36 has the most conserved pKa shifts of any alignment position, suggesting that the role of the conserved Tyr L:37 is electrostatic. The spatial freedom of the residue arises in order to maximize the electrostatic interactions between the tyrosine residue and the substrate.

 
On the other hand, the remaining conserved tyrosine residues are more conformationally restricted and may or may not be able to interact directly with the substrate. In fact, the PDFs clearly demonstrate a spatially conserved distribution of tyrosine residues at 9 and 12 Å, well beyond normal contact cutoffs. Sequence positions being tabulated in these peaks are generally more variable than the other positions directly interacting with the substrate. Strictly sequence-based analyses are unlikely to implicate these positions. Tyrosine identity is conserved at positions L:32, L:36, L:49, H:27 and H:32 63, 76, 74, 58 and 60%, respectively, of the time across a sequence alignment of all solved Ab structures. These conservation quantities are not extraordinary considering that 43% of all sequence positions are conserved better than 60%. Using KabatMan (Martin, 1996Go), we found these results to be consistent with the entire Kabat database. This result indicates that a sequence-only based analysis is unlikely to implicate them as important. Despite the sequence variability in these Tyr hot-spots, the total number of Tyr residues within the combining site is conserved; combining site is defined the same as in Figure 5. The average Tyr occurrence is 7.5 (standard deviation = 1.8). This result occurs because there are many other, less conserved Tyr residues (see Table IV) that can substitute when one of the Tyr hot-spots is mutated. In fact, Tyr pKa shifts in hot-spot positions without consensus are the most common.


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Table IV. Alignment positions with conserved pKa shifts

 
Acidic and basic residues

Interfacial acidic or basic residues are rarely conserved. However, conserved pKa shifts at specific alignment positions are frequently observed. Residue identity at these non-tyrosine hot-spots is indiscriminate. In fact, most are not even conserved enough for a consensus residue to be identified. Analysis of these positions within a sequence alignment of all known antibody structures confirms the generality of this result. Structural scrutiny reveals that residue identity is determined by antigen structural characteristics. Acidic and basic residues tend to arise to satisfy hydrogen bonding, dipole–dipole and salt bridge requirements of the antigen.

Unlike most other non-tyrosine hot-spots, H:94 is highly conserved. An arginine residue occurs 76% of the time. Additionally, a conserved pKa shift occurs in 61% of these arginine residues. The conserved arginine interacts directly with many of the various substrates through hydrogen bonding, dipole–dipole or salt bridge interactions. Like Tyr L:36, Arg H:94 displays significant structural variability. Unlike Tyr L:36, the structure variability includes {alpha}-carbon positions. Tyr L:36 is buried deep in the core and outside the CDR loops. Therefore, conformational changes are generally limited to side-chain torsion angles. Arg H:94 occurs in the third heavy-chain CDR whose structural plasticity allows both main- and side-chain conformational changes. These conformational changes optimize Arg H:94–substrate interactions.

Most of the hapten molecules have various charged groups (i.e. phosphates, carboxylic acids, guanidinium bases and chelated ions). Phosphates are the most common; one-third of the hapten molecules have a phosphate group. No conserved interaction motif is seen between the Ab and phosphate. Despite no particular sequence conservation, cationic combining site residues are always involved in salt bridges with the anionic phosphate. Further, there is little sequence conservation in the remaining acid–base interactions between Ab and hapten. However, most of the charged groups are involved in some sort of stabilizing interaction largely defined by the requirements of the particular antigen.

Antibody design implications

Although it is tempting to view these results in the framework of Ab maturation, we believe that this study has a more direct impact on Ab design. These results suggest that there is a general combining site scaffolding defined by tyrosine and a few other residues (including Arg H:94). Because of conservation, these positions are less attractive from a protein design point of view. On the other hand, Nature frequently optimizes sequences at the pKa shift hot-spots to maximize antigen specificity. It follows that mutant screening (in silico or otherwise) at these positions is more likely to increase substrate specificity than at other positions. These conclusions hold for both anti-protein and anti-hapten Abs.

Conclusions

This study firmly identifies common association motifs across Ab–antigen complexes. The association motif consists of five conserved tyrosine residues and a conserved arginine from CDR-H3. The conserved residues initiate substrate specificity through a host of Ab–antigen interactions and maintain combining site structure. Owing to structural and chemical flexibility, the exact nature of these interactions is not strictly conserved. Substrate specificity is refined by a heterogeneous group of non-covalent Ab–antigen interactions. There is little sequence conservation in these refining interactions; however, they are conserved with alignment ‘hot-spot’ positions. Through a reduction in mutation space, these hot-spots should improve Ab design experimentation.


    Notes
 
2 Present address: Department of Chemistry, California State Polytechnic University, Pomona, 3801 W. Temple Avenue, Pomona, CA 91768, USA Back


    Acknowledgments
 
The authors thank Dr Scott Linthicum for helpful discussions on antibody structure and antigen recognition and Dr Andrew McCammon for providing the UHBD suite of programs, for the calculation of residue pKa values. This work was supported by National Science Foundation Grant DBI 96-04223, National Institutes of Health Grant R01 GM 46535 and a National Center for Supercomputing Applications metacenter computer allocation.


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 Discussion
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Received March 26, 2004; revised June 24, 2003; accepted July 8, 2004.

Edited by Anthony Rees





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