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. 2023 Sep 28;13(1):16281.
doi: 10.1038/s41598-023-42698-7.

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Jiazhi Tang  1 , Cheng Zhang  2 , Nuria Codina Castillo  2 , Christophe J Lalaurie  2 , Xin Gao  3 , Paul A Dalby  4 , Frank Kozielski  5
Affiliations

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Jiazhi Tang et al. Sci Rep. .

Abstract

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Overall A33 Fab structure. The heavy chain is coloured magenta while the light chain is coloured green. A33 Fab illustrates a canonical β-sandwich Ig fold within four domains (VL, VH, CL and CH1) and each domain contains a disulphide bridge between the inner and outer β-sheets (yellow sphere). Missing C-terminal region of the heavy chain (heavyK218 to heavyA228) is marked with a red dash. Image generated using PyMol v2.5: (https://pymol.org/2/).
Figure 2
Figure 2
(a) Superposed Cα trace of A33 Fab structures in space groups P1 (green) and P65 (cyan). The missing regions (heavyS132 to heavyT135 and heavyK218 to heavyA228) are marked with dash lines. The two structures share high similarity but display differences in the switch region. (b) Superposed heavy chain switch region. The triclinic model (P1) is coloured in green while the hexagonal model (P65) is coloured in cyan. Conformational differences can be viewed from heavyS117 to heavyS119 while light chain switch peptides are fixed by a set of hydrogen bonds. The protonated residues have been marked with cross markers. (c) Superposed light chain switch region. Images generated using PyMol v2.5: (https://pymol.org/2/).
Figure 3
Figure 3
CDRs of A33 Fab. (a) CDR1 (brown), CDR2 (green) and CDR3 (black) originating from monoclonal murine A33 Fab are responsible for antigen binding. (b) Superposition of A33 Fab (grey) with Certolizumab (green). Images generated using PyMol v2.5: (https://pymol.org/2/).
Figure 4
Figure 4
Radius of gyration (Rg) of Fab starting from different crystal structures at different pH during the simulation. The Rg of all the five conditions were superimposed in (a) while fewer conditions were plotted separately in (b–d) for the ease of comparison. Six repeats were performed for each unique condition, and the average values were reported with standard error of the mean (SEM) as the error bars.
Figure 5
Figure 5
(a) The average Solvent Accessible Surface Area (SASA) of representative aggregation-prone regions (APR) in the final 20 ns of simulations, in response to various pH from different crystal structures; (b, c) APR SASA of residue lightT31-Y36 during the simulation; (d, e) The SASA of residue lightT129-F139 and residue heavyV173-V188 in the P1, P65 homology models and the MD conditions at time 0;. Error bars are SEM (n = 6 repeats) and are equal for positive and negative values.
Figure 6
Figure 6
The SASA of CL residues lightT129-F139 and corresponding elbow angles during the 80–100 ns (hollow circle) and 0 ns (filled circle). At 0 ns, the P1_pH7_300K data point (green filled) is covered by the P1_pH9_300K one (cyan filled).
Figure 7
Figure 7
Root mean square deviation (RMSD) of Fab starting from different crystal structures at different pH during the simulation. The RMSD of all the 5 conditions were superimposed in (a) while fewer conditions were plotted separately in (b–d) for the ease of comparison. Six repeats were performed for each unique condition, and the average values were reported with standard error of the mean (SEM) as the error bars.
Figure 8
Figure 8
Elbow angle of Fab starting from different crystal structures at different pH during the simulation. The elbow angle of all the 5 conditions were superimposed in (a) while fewer conditions were plotted separately in (b–d) for the ease of comparison. Six repeats were performed for each unique condition, and the average values were reported with standard error of the mean (SEM) as the error bars.
Figure 9
Figure 9
Goodness of fit (Chi-square) between experimental SAXS curves and all frames during Fab MD simulations with different pH and starting crystal structures. Six repeats of each MD condition were plotted in the same colour as previous figures. The top 10 best-fit structures are highlighted as stars to denote their occurrence in simulation time.
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