The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Structural Basis of the Immunological Cross-Reactivity between Kiwi and Birch Pollen.

Allergies related to kiwi consumption have become a growing health concern, with their prevalence on the rise. Many of these allergic reactions are attributed to cross-reactivity, particularly with the major allergen found in birch pollen. This cross-reactivity is associated with proteins belonging to the pathogenesis-related class 10 (PR-10) protein family. In our study, we determined the three-dimensional structures of the two PR-10 proteins in gold and green kiwi fruits, Act c 8 and Act d 8, using nuclear magnetic resonance (NMR) spectroscopy. The structures of both kiwi proteins closely resemble the major birch pollen allergen, Bet v 1, providing a molecular explanation for the observed immunological cross-reactivity between kiwi and birch pollen. Compared to Act d 11, however, a kiwi allergen that shares the same architecture as PR-10 proteins, structural differences are apparent. Moreover, despite both Act c 8 and Act d 8 containing multiple cysteine residues, no disulfide bridges are present within their structures. Instead, all the cysteines are accessible on the protein's surface and exposed to the surrounding solvent, where they are available for reactions with components of the natural food matrix. This structural characteristic sets Act c 8 and Act d 8 apart from other kiwi proteins with a high cysteine content. Furthermore, we demonstrate that pyrogallol, the most abundant phenolic compound found in kiwi, binds into the internal cavities of these two proteins, albeit with low affinity. Our research offers a foundation for further studies aimed at understanding allergic reactions associated with this fruit and exploring how interactions with the natural food matrix might be employed to enhance food safety.

PEP-Patch: Electrostatics in Protein-Protein Recognition, Specificity, and Antibody Developability.

The electrostatic properties of proteins arise from the number and distribution of polar and charged residues. 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 are prerequisites for understanding these processes. Here, we present PEP-Patch, a tool to visualize and quantify the electrostatic potential on the protein surface in terms of surface patches, denoting separated areas of the surface with a common physical property. We highlight its applicability to elucidate protease substrate specificity and antibody-antigen recognition and predict heparin column retention times of antibodies as an indicator of pharmacokinetics.

Structure and Dynamics Guiding Design of Antibody Therapeutics and Vaccines.

Antibodies and other new antibody-like formats have emerged as one of the most rapidly growing classes of biotherapeutic proteins. Understanding the structural features that drive antibody function and, consequently, their molecular recognition is critical for engineering antibodies. Here, we present the structural architecture of conventional IgG antibodies alongside other formats. We emphasize the importance of considering antibodies as conformational ensembles in solution instead of focusing on single-static structures because their functions and properties are strongly governed by their dynamic nature. Thus, in this review, we provide an overview of the unique structural and dynamic characteristics of antibodies with respect to their antigen recognition, biophysical properties, and effector functions. We highlight the numerous technical advances in antibody structure prediction and design, enabled by the vast number of experimentally determined high-quality structures recorded with cryo-EM, NMR, and X-ray crystallography. Lastly, we assess antibody and vaccine design strategies in the context of structure and dynamics.

Grid inhomogeneous solvation theory for cross-solvation in rigid solvents.

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 toward chloroform as a solvent to allow the prediction of passive membrane permeability. Here, we further generalize the GIST algorithm toward all solvents that can be modeled as rigid molecules. This restriction is inherent to the method and is already present in the inhomogeneous solvation theory. Here, we show that our approach can be applied to various solvent molecules by comparing the results of GIST simulations with thermodynamic integration (TI) calculations and experimental results. Additionally, we analyze and compare a matrix consisting of 100 entries of ten 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 0.99 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, which 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.

Corrigendum: Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats.

[This corrects the article DOI: 10.3389/fmolb.2022.812750.].

Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats.

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.

VH -VL interdomain dynamics observed by computer simulations and NMR.

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-1ß 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.