Toward high-resolution homology modeling of antibody Fv regions and application to antibody-antigen docking

Abstract

High-resolution homology models are useful in structure-based protein engineering applications, especially when a crystallographic structure is unavailable. Here, we report the development and implementation of RosettaAntibody, a protocol for homology modeling of antibody variable regions. The protocol combines comparative modeling of canonical complementarity determining region (CDR) loop conformations and de novo loop modeling of CDR H3 conformation with simultaneous optimization of V(L)-V(H) rigid-body orientation and CDR backbone and side-chain conformations. The protocol was tested on a benchmark of 54 antibody crystal structures. The median root mean square deviation (rmsd) of the antigen binding pocket comprised of all the CDR residues was 1.5 A with 80% of the targets having an rmsd lower than 2.0 A. The median backbone heavy atom global rmsd of the CDR H3 loop prediction was 1.6, 1.9, 2.4, 3.1, and 6.0 A for very short (4-6 residues), short (7-9), medium (10-11), long (12-14) and very long (17-22) loops, respectively. When the set of ten top-scoring antibody homology models are used in local ensemble docking to antigen, a moderate-to-high accuracy docking prediction was achieved in seven of fifteen targets. This success in computational docking with high-resolution homology models is encouraging, but challenges still remain in modeling antibody structures for sequences with long H3 loops. This first large-scale antibody-antigen docking study using homology models reveals the level of "functional accuracy" of these structural models toward protein engineering applications.

Figure 1

RosettaAntibody flowchart. The dotted box envelopes the high-resolution CDR loop modeling and V_L-V_H rigid-body minimization steps.

Figure 2

Histogram of local and global loop rmsd of non-H3 CDR loops after comparative modeling for 54 targets. (a) Local rmsd. (b, and c) Global rmsd after grafting of CDR template on (b) the native framework or (c) the BLAST-selected framework.

Figure 3

Global loop rmsd of CDR H3 in native recovery and homology modeling simulations for 54 targets. (a) Lowest scoring and (b) lowest rmsd models from the native recovery simulations. (c) Lowest scoring and (d) lowest rmsd models from homology modeling simulations.

Figure 4

Examples of high, medium and lower quality loop CDR H3 modeling predictions from homology modeling simulations with V_L-V_H rigid-body minimization. Structures are superposed using the C_α atoms of the V_H framework. (a) HyHEL-5 Fab (1jhl), CDR H3 loop length 9, global rmsd 1.1 Å (after superposition of V_H); (b) jel42 Fab (2jel), CDR H3 loop length 9, global rmsd 1.4 Å; (c) antibody 2H1 (2h1p), CDR H3 loop length 11, global rmsd 1.7 Å.

Figure 5

The antigen binding surface of the ten top-scoring homology models superposed on the antibody crystal structure for (a) D3H44 Fab (1jpt) and (b) 4F11E12 Fab (1ynt). The native conformation is colored yellow-green and the low-rmsd model is colored salmon. Side chains are shown for the native and low-rmsd structures for CDR residues that contact the antigen in the co-crystal structure.

Figure 6

Docking energy landscape plots for 4F11E12 Fab complexed with surface antigen SAG1 (1ynt). High-quality decoys (CAPRI criteria44) are indicated by red points (●), medium-quality decoys are indicated by green points (●), acceptable quality decoys are indicated by blue points (●) and incorrect predictions are indicated by open points (○).

Figure 7

Medium accuracy antibody-antigen complex structure predicted for HyHEL-5 antibody complexed with lysozyme (1bql). Docking was performed using the ensemble of the ten low-scoring RosettaAntibody homology models. The antigen is unbound lysozyme (1dkj). The antibody framework regions and the bound antigen are gray, and the docked antigen in the model is green. The CDR loops of the native structure are blue, the non-H3 CDR loops of the homology model antibody are cyan, and the CDR H3 of the homology model antibody is red.