Thermal Stability of Single-Domain-Antibodies Estimated by MD Simulations

Abstract

Antibodies bind to antigens with high specificity and affinity and have complementarity-determining region (CDR) loops, which play an important role in antigen binding. We previously predicted the structure of the CDR-H3 loop by using MD simulations [1] and used MD for the refinement and the ranking of decoys generated by rigid-body antibody-antigen docking [2]. Single-domain-antibodies, sdAbs, function like regular antibodies, however consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets. For applications such as antibody drugs and biosensors, an sdAb with a high thermal stability is required. In this work, we chose seven sdAbs, which have a wide range of Tm-values and have known structures. We then applied MD simulations to estimate their relative stability and compare them with the experimental data. Recently, we executed MD to validate the stability of docking configurations [3] and substrate binding structures [4]. Here, high temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the Tm-values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q-values of hydrophilic residues exhibited an even better correlation. Measuring the Q-value on a per-residue level enabled us to identify residues that contribute significantly to the instability and demonstrate how our analysis can be used in a mutant case-study. [1] H. Nishigami et al. Protein Eng. Des. Sel. 29, 477-484 (2016). [2] N. Shimba et al. J. Chem. Inf. Model. 56, 2005-2012 (2016). [3] G.-J. Bekker et al. J. Chem. Theory Comput. 13, 2389-2399 (2017). [4] N. Numoto et al. Biochemistry 57, 5289-5300 (2018).