Pathogenicity of de novo CACNA1D Ca2+ channel variants predicted from sequence co-variation.

Voltage-gated L-type Cav1.3 Ca2+ channels support numerous physiological functions including neuronal excitability, sinoatrial node pacemaking, hearing, and hormone secretion. De novo missense mutations in the gene of their pore-forming α1-subunit (CACNA1D) induce severe gating defects which lead to autism spectrum disorder and a more severe neurological disorder with and without endocrine symptoms. The number of CACNA1D variants reported is constantly rising, but their pathogenic potential often remains unclear, which complicates clinical decision-making. Since functional tests are time-consuming and not always available, bioinformatic tools further improving pathogenicity potential prediction of novel variants are needed. Here we employed evolutionary analysis considering sequences of the Cav1.3 α1-subunit throughout the animal kingdom to predict the pathogenicity of human disease-associated CACNA1D missense variants. Co-variation analyses of evolutionary information revealed residue-residue couplings and allowed to generate a score, which correctly predicted previously identified pathogenic variants, supported pathogenicity in variants previously classified as likely pathogenic and even led to the re-classification or re-examination of 18 out of 80 variants previously assessed with clinical and electrophysiological data. Based on the prediction score, we electrophysiologically tested one variant (V584I) and found significant gating changes associated with pathogenic risks. Thus, our co-variation model represents a valuable addition to complement the assessment of the pathogenicity of CACNA1D variants completely independent of clinical diagnoses, electrophysiology, structural or biophysical considerations, and solely based on evolutionary analyses.

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.

Asymmetric contribution of a selectivity filter gate in triggering inactivation of CaV1.3 channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition.

Determining internal coordinate sets for optimal representation of molecular vibration.

Arising from the harmonic approximation in solving the vibrational Schrödinger equation, normal modes dissect molecular vibrations into distinct degrees of freedom. Normal modes are widely used as they give rise to descriptive vibrational notations and are convenient for expanding anharmonic potential energy surfaces as an alternative to higher-order Taylor series representations. Usually, normal modes are expressed in Cartesian coordinates, which bears drawbacks that can be overcome by switching to internal coordinates. Considering vibrational notations, normal modes with delocalized characters are difficult to denote, but internal coordinates offer a route to clearer notations. Based on the Hessian, normal mode decomposition schemes for a given set of internal coordinates can describe a normal mode by its contributions from internal coordinates. However, choosing a set of internal coordinates is not straightforward. While the Hessian provides unique sets of normal modes, various internal coordinate sets are possible for a given system. In the present work, we employ a normal mode decomposition scheme to choose an optimal set. Therefore, we screen reasonable sets based on topology and symmetry considerations and rely on a metric that minimizes coupling between internal coordinates. Ultimately, the Nomodeco toolkit presented here generates internal coordinate sets to find an optimal set for representing molecular vibrations. The resulting contribution tables can be used to clarify vibrational notations. We test our scheme on small to mid-sized molecules, showing how the space of definable internal coordinate sets can significantly be reduced.

Addressing Challenges of Macrocyclic Conformational Sampling in Polar and Apolar Solvents: Lessons for Chameleonicity.

We evaluated a workflow to reliably sample the conformational space of a set of 47 peptidic macrocycles. Starting from SMILES strings, we use accelerated molecular dynamics simulations to overcome high energy barriers, in particular, the cis-trans isomerization of peptide bonds. We find that our approach performs very well in polar solvents like water and dimethyl sulfoxide. Interestingly, the protonation state of a secondary amine in the ring only slightly influences the conformational ensembles of our test systems. For several of the macrocycles, determining the conformational distribution in chloroform turns out to be considerably more challenging. Especially, the choice of partial charges crucially influences the ensembles in chloroform. We address these challenges by modifying initial structures and the choice of partial charges. Our results suggest that special care has to be taken to understand the configurational distribution in apolar solvents, which is a key step toward a reliable prediction of membrane permeation of macrocycles and their chameleonic properties.

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.

Asymmetric Contribution of a Selectivity Filter Gate in Triggering Inactivation of CaV1.3 Channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultra-highly conserved tryptophan residues in the inter-domain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudo-tetrameric CaV pore domain for feedback inhibition.

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.

Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine.

The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates thein vitrobioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. Thisin vitroapproach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation with and without limonene, giving a kinetic constant of the reaction,KM, of 103µM and 94.1µM, respectively, and a maximum reaction rate,Vmax, of 46.8 pmol min-1106cells-1and 56.0 pmol min-1106cells-1with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half-maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413µM.

Cross-linking disulfide bonds govern solution structures of diabodies.

In the last years, antibodies have emerged as a promising new class of therapeutics, due to their combination of high specificity with long serum half-life and low risk of side-effects. Diabodies are a popular novel antibody format, consisting of two Fv domains connected with short linkers. Like IgG antibodies, they simultaneously bind two target proteins. However, they offer altered properties, given their smaller size and higher rigidity. In this study, we conducted the-to our knowledge-first molecular dynamics (MD) simulations of diabodies and find a surprisingly high conformational flexibility in the relative orientation of the two Fv domains. We observe rigidifying effects through the introduction of disulfide bonds in the Fv -Fv interface and characterize the effect of different disulfide bond locations on the conformation. Additionally, we compare VH -VL orientations and paratope dynamics between diabodies and an antigen binding fragment (Fab) of the same sequence. We find mostly consistent structures and dynamics, indicating similar antigen binding properties. The most significant differences can be found within the CDR-H2 loop dynamics. Of all CDR loops, the CDR-H2 is located closest to the artificial Fv -Fv interface. All examined diabodies show similar VH -VL orientations, Fv -Fv packing and CDR loop conformations. However, the variant with a P14C-K64C disulfide bond differs most from the Fab in our measures, including the CDR-H3 loop conformational ensemble. This suggests altered antigen binding properties and underlines the need for careful validation of the disulfide bond locations in diabodies.

High Intrinsic Oncogenic Potential in the Myc-Box-Deficient Hydra Myc3 Protein.

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver.

Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9.

A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the minor (NPNV) repeat domain, which is stabilized by a unique set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts PfCSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum.

Vibrational Predissociation Spectra of C2 N- and C3 N- : Bending and Stretching Vibrations.

We present infrared predissociation spectra of C2 N- (H2 ) and C 3 N- (H2 ) in the 300-1850 cm-1 range. Measurements were performed using the FELion cryogenic ion trap end user station at the Free Electron Lasers for Infrared eXperiments (FELIX) laboratory. For C2 N- (H2 ), we detected the CCN bending and CC-N stretching vibrations. For the C3 N- (H2 ) system, we detected the CCN bending, the CC-CN stretching, and multiple overtones and/or combination bands. The assignment and interpretation of the presented experimental spectra is validated by calculations of anharmonic spectra within the vibrational configuration interaction (VCI) approach, based on potential energy surfaces calculated at explicitly correlated coupled cluster theory (CCSD(T)-F12/cc-pVTZ-F12). The H2 tag acts as an innocent spectator, not significantly affecting the C2,3 N- bending and stretching mode positions. The recorded infrared predissociation spectra can thus be used as a proxy for the vibrational spectra of the bare anions.

Evolution of the Immunoglobulin Isotypes-Variations of Biophysical Properties among Animal Classes.

The adaptive immune system arose around 500 million years ago in jawed fish, and, since then, it has mediated the immune defense against pathogens in all vertebrates. Antibodies play a central role in the immune reaction, recognizing and attacking external invaders. During the evolutionary process, several immunoglobulin isotypes emerged, each having a characteristic structural organization and dedicated function. In this work, we investigate the evolution of the immunoglobulin isotypes, in order to highlight the relevant features that were preserved over time and the parts that, instead, mutated. The residues that are coupled in the evolution process are often involved in intra- or interdomain interactions, meaning that they are fundamental to maintaining the immunoglobulin fold and to ensuring interactions with other domains. The explosive growth of available sequences allows us to point out the evolutionary conserved residues and compare the biophysical properties among different animal classes and isotypes. Our study offers a general overview of the evolution of immunoglobulin isotypes and advances the knowledge of their characteristic biophysical properties, as a first step in guiding protein design from evolution.

Structural mechanism of Fab domain dissociation as a measure of interface stability.

Therapeutic antibodies should not only recognize antigens specifically, but also need to be free from developability issues, such as poor stability. Thus, the mechanistic understanding and characterization of stability are critical determinants for rational antibody design. In this study, we use molecular dynamics simulations to investigate the melting process of 16 antigen binding fragments (Fabs). We describe the Fab dissociation mechanisms, showing a separation in the VH-VL and in the CH1-CL domains. We found that the depths of the minima in the free energy curve, corresponding to the bound states, correlate with the experimentally determined melting temperatures. Additionally, we provide a detailed structural description of the dissociation mechanism and identify key interactions in the CDR loops and in the CH1-CL interface that contribute to stabilization. The dissociation of the VH-VL or CH1-CL domains can be represented by conformational changes in the bend angles between the domains. Our findings elucidate the melting process of antigen binding fragments and highlight critical residues in both the variable and constant domains, which are also strongly germline dependent. Thus, our proposed mechanisms have broad implications in the development and design of new and more stable antigen binding fragments.

Challenges in antibody structure prediction.

Advances in structural biology and the exponential increase in the amount of high-quality experimental structural data available in the Protein Data Bank has motivated numerous studies to tackle the grand challenge of predicting protein structures. In 2020 AlphaFold2 revolutionized the field using a combination of artificial intelligence and the evolutionary information contained in multiple sequence alignments. Antibodies are one of the most important classes of biotherapeutic proteins. Accurate structure models are a prerequisite to advance biophysical property predictions and consequently antibody design. Specialized tools used to predict antibody structures based on different principles have profited from current advances in protein structure prediction based on artificial intelligence. Here, we emphasize the importance of reliable protein structure models and highlight the enormous advances in the field, but we also aim to increase awareness that protein structure models, and in particular antibody models, may suffer from structural inaccuracies, namely incorrect cis-amide bonds, wrong stereochemistry or clashes. We show that these inaccuracies affect biophysical property predictions such as surface hydrophobicity. Thus, we stress the importance of carefully reviewing protein structure models before investing further computing power and setting up experiments. To facilitate the assessment of model quality, we provide a tool "TopModel" to validate structure models.

Structural Characterization of Nanobodies during Germline Maturation.

Camelid heavy-chain antibody variable domains (VHH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VHH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VHH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VHH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.

Assessing developability early in the discovery process for novel biologics.

Beyond potency, a good developability profile is a key attribute of a biological drug. Selecting and screening for such attributes early in the drug development process can save resources and avoid costly late-stage failures. Here, we review some of the most important developability properties that can be assessed early on for biologics. These include the influence of the source of the biologic, its biophysical and pharmacokinetic properties, and how well it can be expressed recombinantly. We furthermore present in silico, in vitro, and in vivo methods and techniques that can be exploited at different stages of the discovery process to identify molecules with liabilities and thereby facilitate the selection of the most optimal drug leads. Finally, we reflect on the most relevant developability parameters for injectable versus orally delivered biologics and provide an outlook toward what general trends are expected to rise in the development of biologics.

Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1.

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.