Do antibody CDR loops change conformation upon binding?

Abstract

Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.

Keywords: CDR flexibility; CDRs; antibodies; antibody binding; antibody structure; complementarity determining regions.

Figure 1.

Filtering AbDb files. Starting from the bottom, we eliminate structures with resolution worse than 2.8Å and retain antibodies (Abs) that have both heavy and light variable domains, then eliminate files with missing residues in any of the six CDRs, where the Cα atom B-factor is missing (i.e. 0) or > 80, or a cis non-proline residue is present in an unbound antibody, leading to 364 Abs with 1109 entries. We then retained antibodies with both unbound and bound structures (181 Abs with 773 entries) and performed global and local fitting. Finally, we eliminated unbound/bound structure pairs whose framework region showed ≥1.0Å global Cα RMSD to minimize the impact of the framework region on CDR conformational change and followed by rechecking that both unbound and bound structures are available for an antibody, which led to the elimination of four antibodies. This led to a final set of 749 entries representing 177 antibodies. See supplementary file Supp01_unbound_and_bound_abs.xlsx for the initial dataset of entries with both bound and unbound structures from AbDb. See supplementary file Supp04_antibody_filtering.xlsx for information on entries retained and rejected at each step.

Figure 2.

Filtering AbDb unbound structures. Filtering steps for unbound antibody structures uses the same protocol as in Figure 1. The numbers of entries and antibodies retained at each step are indicated. See supplementary file Supp05_unbound_filtering.xlsx for information on entries retained and rejected at each step.

Figure 3.

Conformational change types. The two major canonical clusters (i.e. sub-graphs) of the LRC group ‘H2–10-allT’ are shown and are denoted as c₁ and c₂ (area shaded in gray). Each canonical cluster consists of AP clusters (i.e., nodes in blue and orange). The AP cluster of the unbound conformer is labeled as a_u, and its canonical cluster is denoted as c_u, in this case, c₁. The AP cluster and canonical cluster of a bound conformation x are denoted as a_B and c_B on the graph. Comparing a_B,c_B with a_u,c_u, we can define four types of CDR conformational change upon binding: (1) identical AP cluster: the bound conformer is merged with the same AP cluster as the unbound. (2) AP-cluster shift: the bound conformer is merged with a different AP cluster in the same canonical cluster as the unbound. (3) canonical-cluster shift: the bound conformer is merged with an AP cluster in a canonical cluster different from the unbound; (4) non-canonical conformation: x is not merged with any AP cluster or canonical cluster. See Table 1.

Figure 4.

CDR conformational change distribution from global and local fitting. Distributions of conformational change (measured as Cα RMSD) from global fitting and local fitting. Each box represents the first quartile, median and the third quartile while the whiskers represent the lower and upper fence (Q3 + 1.5×IQR meaning 3rd quartile plus one and half inter-quartile range). Outliers are shown as circles above the upper fence. To assist comparison, the Cα RMSD at 0.5Å and 1.0Å are plotted as dashed lines. A p-value to compare local and global fitting was calculated using a two-sample Mann-Whitney U test. In all cases, **** indicates p ≤0.0001 indicating that global and local fitting are significantly different for all CDRs.

Figure 5.

CDR loop movement upon complexation against loop length. The global Cα RMSD of each CDR upon binding (Cα RMSD) versus loop length (number of residues) are plotted as boxplots, with outliers (exceeding upper fence values Q3 + 1.5×IQR) shown as circles. A horizontal dashed line is drawn at 1.0Å Cα RMSD on each box plot. The number of CDR loops of each loop length are also plotted as histograms.

Figure 6.

LRC groups. Each subplot shows the number of entries in each LRC group. For CDR-H3, only groups with more than 10 entries are shown.

Figure 7.

Structure clusters of predominant non-CDR-H3 CDR LRC groups. Subplot titles are CDR LRC group names, and the percentage given in parenthesis denotes the ratio of the group size (number of AbDb entries) to the entire set (1091 entries). Each node represents an AP cluster which consists of a set of similar CDR structures and from which a representative structure (also called an exemplar structure) was identified. The edges between pairs of nodes indicate the exemplar structures of both nodes are similar following our criteria under Cartesian space and thus belong to the same canonical cluster. Nodes, directly or indirectly connected, are given the same color. The major groups are colored in black and smaller ones in gray. Note we use edges to indicate connectivity only, which means the distance between a pair of nodes is trivial in this case. The placing of nodes in the figure is purely illustrative.

Figure 8.

Local Cα RMSD of antibodies of each conformational change type. Each subplot is a kernel density estimation of the local Cα RMSD of unbound/bound CDR conformation pairs found in each conformational change type. The number in each subplot parenthesis indicates number of antibodies.

Figure 9.

Example of each conformational change type. The figure shows one locally fitted CDR-H3 unbound/bound pair for each conformational change type. The bound CDR-H3 loop is colored black and the unbound loop is gray. (a) Non-canonical cluster conformation: unbound (6umh_0) and bound (6umg_0P), global Cα RMSD of 8.41Å; (b) Canonical-cluster shift: unbound (7n3g_0) and bound (7n3i_0P), global Cα RMSD of 5.28Å; (c) AP-cluster shift: unbound (1kcv_0) and bound (1kcs_0P), global Cα RMSD of 2.38Å; (d) and (e) identical-AP cluster — (d) is locally fitted and (e) is globally fitted to show the loop ‘flapping’ effect. (d) global Cα RMSD 2.27Å, (e) global Cα RMSD 0.54Å.

Figure 10.

Impact of antigen mutations on CDR conformation. CDRs within clusters of identical antibodies binding to multiple antigens were locally fitted with one another and the Cα RMSDs were plotted. Where, within this set, there was more than one example of the same antibody binding to the same antigen, an exemplar was selected.