Conformational Dynamics of Human IgG1 Antibody

RMSD, RMSF and Rg were computed on C-alpha atoms by MDTraj^{38 (link)}. The analysis of cMD was performed on the concatenated replicas of each system excluding the first 300 ns that were considered as equilibration steps. The movement of Fab domains was described by means of ϕ (longitude) and θ (latitude) angles defined in a reference frame jointed to Fc and centered in the hinge with axes defined as follows: z axis collinear to Fc and directed toward Fabs, x axis parallel to a vector joining mid-Fc (CH2 regions) and y axis defined by right hand rule. For a more detailed description see the work by Saporiti et al.^{20 (link)} An arbitrary threshold of 85° was chosen for θ angle to discriminate between Y- and T-shaped conformations. Specifically, we considered that the only one fully crystalized human IgG1 (PDB ID: 1HZH)^{31 (link)}, that is classified as a T-shaped conformation^{39 (link)}, presents θ > 90° for both Fab domains, and we took into account also the conformational variability expected from MD simulations. The distance between the CH2 domains was measured between the glycosylated Asn using MDTraj^{38 (link)}. Then, box plots were produced to evaluate the statistical significance of the observed values in the total 21,000 frames. For the aMD, a reweighing procedure was applied according to methods described by Miao et al.^{40 (link)} using Maclaurin expansion to the 10th order to approximate the free energy surface of the system as a function of θ angles. The RMSD matrices for the cluster analysis (of both cMD and aMD) were generated with CPPTRAJ^{41 (link)}, while the clusters were obtained using a customized script based on the GROMOS algorithm^{42 (link)}. In the case of antibodies C-alpha atoms were considered for the analysis, while for glycans the oxygens involved in glycosidic bonds. RMSD-threshold of 7.5 Å and 6.5 Å were used for the antibodies in cMD and aMD, respectively, and the maximum number of clusters was set to 15 and 10, respectively. For glycans clustering the RMSD-threshold was set to 1 Å and the maximum number of clusters to 10. The essential dynamics (ED) was computed on the overall trajectories by the covariance analysis tool of GROMACS 2020.1^{20 (link),43 (link)}. Then, the resulting trajectories, projected along the first and the second eigenvectors, were filtered by the frames included in the energy minimum that was identified from the FES (computed as function of θ angles) and were used to calculate the Δϕ distribution. The minimum distance between glycan chains was computed by CPPTRAJ^{41 (link)} and the “nativecontacts” tool with the “mindist” option, while the distance between the center of mass of each chain and itself was computed with the “distance” tool. For the latter, the trajectories were pre-aligned on the Fc. The contacts between LCs and the hinge region were computed by CPPTRAJ^{41 (link)} with the “nativecontacts” tool, considering heavy atoms and a threshold distance of 4 Å. The hydrogen bonds (H-bonds) analysis was computed by a customized python script based on the MDTraj H-bonds identification tool^{20 (link)}.