Improving Loop Modeling of the Antibody Complementarity-Determining Region 3 Using Knowledge-Based Restraints

<div><p>Structural restrictions are present even in the most sequence diverse portions of antibodies, the complementary determining region (CDR) loops. Previous studies identified robust rules that define canonical structures for five of the six CDR loops, however the heavy chain CDR 3 (HCDR3) defies standard classification attempts. The HCDR3 loop can be subdivided into two domains referred to as the “torso” and the “head” domains and two major families of canonical torso structures have been identified; the more prevalent “bulged” and less frequent “non-bulged” torsos. In the present study, we found that Rosetta loop modeling of 28 benchmark bulged HCDR3 loops is improved with knowledge-based structural restraints developed from available antibody crystal structures in the PDB. These restraints restrict the sampling space Rosetta searches in the torso domain, limiting the φ and ψ angles of these residues to conformations that have been experimentally observed. The application of these restraints in Rosetta result in more native-like structure sampling and improved score-based differentiation of native-like HCDR3 models, significantly improving our ability to model antibody HCDR3 loops.</p></div

Bulged and non-bulged dihedral angle measurements.

<p>Bulged and non-bulged dihedral angle measurements.</p

Bulged torso restraints improve native-like HCDR3 sampling and recovery.

<p>Using Rosetta LoopModel, 1,000 models of the benchmark antibody 4G5Z (circles) were generated with or without bulged restraints and these models were then scored with or without bulged restraints (panel A, modeled and scored without restraints; panel B, modeled without but scored with restraints; panel C, modeled with but scored without restraints; panel D, modeled and scored with restraints). The native crystal structure 4G5Z was also minimized using Rosetta FastRelax, generating 20 structures (black x’s). The total HCDR3 score (in Rosetta Energy Units, or REU) is shown versus the Cα root mean square deviation of the HCDR3 loop, normalized to that of a protein loop containing 16 residues (RMSD16, in Å) to the native crystal structure. Models with scores ranked in the top 10% and RMSD16 ≤ 2 Å have been colored blue, while models with scores ranked below the top 10% and RMSD16 > 2 Å have been colored red. Improved native-like HCDR3 sampling is observed as a greater density of low RMSD16 models (blue circles) in comparison to Panel A, while improved model recovery is defined as a greater correlation between RMSD16 and score (colored vs. gray circles) in comparison to Panel A, as seen in panels C and D.</p

Torso restraints improve recovery of native-like bulged HCDR3 loops.

<p>For each benchmark antibody structure, 1,000 models were generated with or without bulged torso restraints. The number of models below 2 Å RMSD16 to the native structure, best RMSD16 sampled, average RMSD16 of the best 10 models ranked by RMSD16, RMSD16 of the best model ranked by Rosetta score, average RMSD16 of the top 10 models ranked by Rosetta score, and the rank of the first model below 2 Å when sorted by Rosetta score are provided. For RMSD16-containing cells, blue shading represents RMSD16 ≤ 1 Å; yellow shading represents RMSD16 between 1 and 2 Å; red represents RMSD16 > 2 Å. For rank-containing cells, blue shading represents rank 1; yellow shading represents ranks 2 to 10; red shading represents ranks > 10.</p

Torso restraints improve sampling of bulged HCDR3 loops.

<p>For each benchmark antibody structure, 1,000 models were generated with or without bulged torso restraints. The number of models below 2 Å RMSD16 to the native structure, the best RMSD16 sampled, and the average RMSD16 of the best 10 models ranked by RMSD16 are provided. For RMSD16-containing cells, blue shading represents RMSD16 ≤ 1 Å; yellow shading represents RMSD16 between 1 and 2 Å; red represents RMSD16 > 2 Å. For cells containing the number of models below 2 Å, blue shading represents ≥ 100 models; yellow shading represents ≥ 10 models; red shading represents fewer than 10 models.</p

Experimentally derived antibodies used to benchmark bulged torso restraints.

<p>Experimentally derived antibodies used to benchmark bulged torso restraints.</p

Cluster analysis of bulged HCDR3 loop modeling.

<p>Calibur was used to cluster the 1,000 models generated with or without bulged torso restraints for each antibody, using a threshold of 2.0. Clusters containing less than 1% of the total models were omitted from analysis; models generated for benchmark antibodies 4F58, 1HZH, 4LKC, 1RHH and 4FNL did not produce any large clusters upon analysis (N/A). Average Rosetta score was calculated for each cluster, and the cluster with the lowest average score was selected as the “correct” cluster. The size of this correct cluster (and it’s rank among cluster sizes), its average RMSD16 to the native structure (and rank among average RMSD16 measurements) are provided. Cells containing rank data are shaded blue if the value represents the top rank, yellow for ranks 2–3, and red for ranks >3; if only one cluster (1*) was found, the cell is shaded gray. For RMSD16-containing cells, blue shading represents RMSD16 ≤ 1 Å; yellow shading represents RMSD16 between 1 and 2 Å; red represents RMSD16 > 2 Å. Values were omitted from column averages if ≤1 cluster was found.</p