The relative orientation of the two variable domains, VH and VL, influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH‐VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single‐chain variable fragment antibody (scFv) in complex with IL‐1β and an antigen‐binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1‐10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH‐VL interdomain variations comparable to the distributions observed for known X‐ray structures of antibodies. Significance Statement Antibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen‐binding fragment (Fab), in particular the variable fragment region (Fv). Antigen‐binding is mediated by the complementarity‐determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL. The relative orientation of the VH and VL domains influences the shape of the antigen‐binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.

As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.

Grid Inhomogeneous Solvation Theory, (GIST), has proven useful to calculate localized thermodynamic properties of water around a solute. Numerous studies have leveraged this information to enhance structure-based binding predictions. We have recently extended GIST towards chloroform as a solvent to allow prediction of passive membrane permeability. Here, we further generalize the GIST algorithm towards all solvents that can be modeled as rigid molecules. This restriction is inherent to the method and is already present in Inhomogeneous Solvation Theory (IST). Here, we show that our approach can be applied to various solvent molecules, by comparing the results of GIST simulations with TI calculations and experimental results. Additionally, we analyze and compare a matrix consisting of 100 entries of 10 different solvent molecules solvated within each other. We find that the GIST results are highly correlated with TI calculations as well as experiments. For some solvents, we find Pearson correlations of up to 0.97 to the true entropy, while others are affected by the first-order approximation more strongly. The enthalpy-entropy splitting provided by GIST allows us to extend a recently published approach, that estimates higher order entropies by a linear scaling of the first-order entropy, to solvents other than water. Furthermore, we investigate the convergence of GIST in different solvents. We conclude that our extension to GIST reliably calculates localized thermodynamic properties for different solvents and thereby significantly extends the applicability of this widely used method.

The electrostatic properties of proteins arise from the number and distribution of polar and charged residues. Due to their long-ranged nature, electrostatic interactions in proteins play a critical role in numerous processes, such as molecular recognition, protein solubility, viscosity, and antibody developability. Thus, characterizing and quantifying electrostatic properties of a protein is a pre-requisite for understanding these processes. Here, we present PEP-Patch, a tool to visualize and quantify the electrostatic potential on the protein surface and showcase its applicability to elucidate protease substrate specificity, antibody-antigen recognition and predict heparin column retention times of antibodies as an indicator of pharmacokinetics.