Thermal stability of single-domain antibodies estimated by molecular dynamics simulations

Abstract

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 and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m ) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High-temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per-residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.

Keywords: melting temperature; molecular dynamics simulation; point mutations; single-domain antibody; thermal stability.

Figure 1

(A) Multiple sequence alignment of 4idl, 1fvc, 4w70, 1mel, 5sv4, 3b9v, and 4tyu performed by Clustal Omega,13 where the consensus symbols shown below the alignment and the residue colors are the defaults used by Clustal Omega. The location of the CDR loops is indicated in the figure. (B) Superposition of 4idl, 1fvc, 4w70, 1mel, 5sv4, 3b9v, and 4tyu with the CDR3 loop in red, black, orange, blue, cyan, green, and magenta, respectively. The image was drawn by Molmil,14 a WebGL molecular viewer developed by Protein Data Bank Japan.15, 16

Figure 2

(A) Average Q value over the final 30 ns with standard deviation against the experimental T _m per simulation temperature (300 K, 400 K, and 500 K). The data for 4idl, 1fvc, 4w70, 1mel, 5sv4, 3b9v, and 4tyu are shown in red circle, black upper triangle, orange leftward triangle, blue square, cyan rightward triangle, green lower triangle, and magenta star, respectively, with error bars. (B) Pearson correlation coefficient (r) of each average Q value of the described group pairs to the experimental T _m for different group combinations and temperatures (300 K, 400 K, and 500 K). The hydrophilic‐all group is the average Q value between the hydrophilic residues (Asp, Glu, Gln, Asn, Arg, Lys, and His) versus all residues, the all–all group is the regular average Q value and the hydrophobic‐small group is the average Q value between the hydrophobic (Phe, Tyr, Trp, Leu, Val, Ile, Met, Cys, and Pro) versus the small (Gly, Ala, Ser, and Thr) residues. (C) Average Q value over the final 30 ns with standard deviation against the experimental T _m per group pair [top 3 in Fig. 2(B)] for the 400 K simulations.