Figure 1.
Multi-state and single-state design methodology.
For a simplified view of the methodology, only the complexes from inferred germline VH5-51 are presented. (A) The heavy chain variable segment amino acid sequences taken from the PDB were aligned. Position candidates were chosen for design if the position differed from the germline sequence in at least one mature complex. (B) Co-crystal structures for each VH5-51 derived complex are shown with heavy chain in black, light chain in grey, antigen in magenta, and designed positions highlighted in gold. Single- and multi-state design schemes are shown where each complex was designed alone (single-state) where the designed positions were optimized to minimize the energy for a single antigen target, or a minimized energy for all complexes considered together (multi-state) where the sequence returned was an energetic consensus among each complex considered. (C) Sequence logos were generated to show 100 design models. Each position in the sequence logo corresponds to a position conserved for design. The sequence logos then were compared to the mature or germline sequence for each antibody. (D) Bit-scores were determined quantitatively by measuring the frequency of a letter at each position. The bit-score measures the designed residues compared to either the germline sequence or the mature sequence. The normalization factor was the [total bit-score]/[perfect design score](see Methods).
Table 1.
Antibody-antigen test set complexes.
Figure 2.
Multi-state designs toward the germline sequence, single-state to mature sequences.
Antibodies encoded by the same inferred germline VH gene preferred germline sequences when considered in the multi-state design, inferring a more flexible combining site. (A) The bar graph shows the bit-score for each of the three different inferred germline groups and then the sum of the scores in a grouped bar. A perfect design would have a normalized bit-score of 1.0, and summated score of 3.0 for three germline groups. Multi-state design preferred germline sequences for all complexes, while in contrast single-state design preferred mature sequences (p<0.0001). (B) The change in bit-score is determined to be the proclivity to either the mature (positive score) or the germline (negative score) sequence. Each complex was assigned a change in bit-score. The change in proclivity between design protocols was significant (p<0.0001). (C) Each complex was scored against mature and germline sequences and a difference was calculated (Δbit-score). Positive numbers returned showed a proclivity towards mature sequences, while a negative score suggested a design toward germline. A tight correlation was observed (r2 = 0.8263) for the in silico predicted optimization for specificity versus polyspecificity (Δbit-score) and the in vivo maturation process (plotted as the mutation percentage away from VH gene sequence).
Figure 3.
Phi-psi variances for framework residues.
The degree of structural variation of the framework residues were measured as the standard deviation of the phi and psi angles over each residue position. (A) Side view of immunoglobulin fold for VH5-51 complexes aligned by framework residues. Beta-sheets included in the analysis are shown as a cartoon representation, while loop regions are in a transparent ribbon representation. Framework 1 is shown in brown, CDR 1 in green, framework 2 in black, CDR 2 in magenta, and framework 3 in cyan. (B) Top down view. (C) The standard deviations of the phi-psi angles of each framework position were binned into either a residue that was found to be critical for polyspecificity (recovered to germline) or a residue that was not recovered to germline in multi-state design. For each position, the phi-psi angles were averaged, and the standard error of the mean was calculated. An average of 19.6°±2.0° for germline recovered residues and 15.47°±1.5° for non-germline recovered residues supporting our hypothesis that residues which enable polyspecificity alter beta-sheet packing to a greater degree than residues that do not. The axis is normalized to 18.7°±0.9°, the average deviation for all beta-sheet framework positions.
Figure 4.
Modified Colliers de Perles representation of VH gene segments.
The 98 amino acids present in VH 1-69, VH3-23, or VH5-51 are shown in a Collier de Perles two-dimensional representation and numbered according to the IMGT numbering scheme [40]. Hatched circles are missing residues according to the IMGT numbering scheme and are shown to make graphs consistent. Square boxes represent the boundary between framework and CDR loops. The anti-parallel beta-sheets are represented A–F. A dashed line is shown that divides interface residues with residues that were found to be outside of the interface in a majority of the cases. Interface residues are colored with a blue-pink gradient indicating a numerical antigen contact score defined by a change in neighbors between the free and bound complex (see Methods). Non-interface residues are colored with a green-orange gradient according to their degree of burial defined through a neighbor count. Residues that are transparent were not considered for redesign as they were conserved across all complexes considered. (A, B, C) show the germline sequence represented in the immunoglobulin fold with the thickness of each line representing the design bit-score for that position relative to the germline sequence for multi-state design protocols for VH1-69, VH3-23, or VH5-51, respectively. (D, E, F) show the germline sequence represented, but the thickness of the line corresponds to the mature sequence bit-score averaged over each complex for the single-state design protocol for VH1-69, VH3-23, or VH5-51, respectively.
Figure 5.
Interface occurrences affect germline sequence recovery.
For VH3-23 (A) and VH1-69 complexes (B), we binned each residue position into how many times it occurred in an interface (interface ensembles). Most designed positions never occurred in an interface. As their occurrences became more frequent, we observed a trend for increasing the recovered germline residue. This trend fell off for VH1-69 complexes (B) for positional occurrences between 5–8 interfaces.
Figure 6.
Rosetta multi-state design solutions for non-germline amino acids represent incomplete sampling for VH5-51 complexes.
We evaluated a complete germline reversion of VH5-51 sequence versus the sequences output by multi-state design. (A) Consideration of positions in which the multi-state design algorithm chose a non-germline amino acid for at least 10% of the models where evaluated. The difference in energy of the germline sequence and the multi-state design solution sequence is shown for each position. Bars above 0 represent the multi-state design sequence preferred while bars below the line represent the germline amino acid preference. The horizontal dashed line at 0.7 Rosetta energy units (REU) shows the average energy difference between the germline and mature sequence and is represented as a marker for sequence tolerance. (B) The multiple sequence alignment for each VH5-51 complex is shown and compared with the germline sequence. Sequences highlighted in bold were considered for design. Sequences highlighted in green are positions in which the multi-state design algorithm chose the germline amino acid as the design solution. The numbers in the bottom row are the alignment-numbering scheme of each position and correspond to the position numbers in (A).