Multi-constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility

Abstract

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen-binding site can significantly enlarge the repertoire. Among the six complementarity determining regions (CDRs) that generally comprise the binding site, the CDR H3 loop is particularly variable. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi-constraint computational design strategy, along with the Rosetta scoring function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen-bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by preorganizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity.

Figure 1

Computational strategy for estimating the degree of optimization of the native sequence for conformational flexibility. The CDR H3 loop of germline antibodies is expected to show higher native sequence recovery in multi-constraint simulations (case 2) than in each of the single-constraint simulations (case 1).

Figure 2

Superimposition of the V_H domain of the germline 7g12 antibody. Bound (pdb: 1n7m) and free (pdb: 1ngz) forms are shown in green and magenta, respectively.

Figure 3

Average native sequence recovery for CDR H3 loops in germline and mature antibodies. The following design simulations were performed: single-constraint design for the bound conformation (white bar), the free conformation (grey bar) and multi-constraint design for both conformations (black bar) for germline and mature antibodies crystallized in different bound and free conformations (Table I). The star indicates that there is a statistically significant difference (as determined by a Binomial test) between the native sequence recovery obtained from multi- and single-constraint design simulations for germline antibodies.

Figure 4

Average native sequence recovery for CDR H3 loops in germline antibodies and their corresponding mature forms. The single-constrain design strategy was used in all cases. Note that for this analysis we grouped as germline the antibody state with no or short-term exposure to antigen (which was omitted in all simulations) and as mature the corresponding antibody after longer term exposure to the same antigen. In each case, a corresponding pair consists of structures that are both either bound to the same epitope or free (Table III). The star indicates that the observed difference is statistically significant (as determined by a Binomial test).

Figure 5

Substitution of Asn by Phe in position 102 improves atomic packing in the d44.1 antibody. The CDR H3 residue 102 and its neighbors (all heavy atoms within 5Å of the residue 102 side chain) are shown in space-fill representation. Residues 46, 49 and 50 belong to the light chain and residues 100,102 and 104 belong to the heavy chain CDR H3 loop. Panels A, B, C correspond to the native d44.1 antibody (pdb: 1mlb), a model of the designed d44.1 antibody and native f10.6.6 antibody (pdb: 2q76); (note that the NZ atom of Lys 49 is not seen in panel A because is located further than 5Å from the Asn 102 side chain).