Muscular Strategies for Correcting the Pelvic Position to Improve Posture-An Exploratory Study.

The correction of postural weaknesses through the better positioning of the pelvis is an important approach in sports therapy and physiotherapy. The pelvic position in the sagittal plane is largely dependent on the muscular balance of the ventral and dorsal muscle groups. The aim of this exploratory study was to examine whether healthy persons use similar muscular activation patterns to correct their pelvic position or whether there are different motor strategies. The following muscles were recorded in 41 persons using surface electromyography (EMG): M. trapezius pars ascendens, M. erector spinae pars lumbalis, M. gluteus maximus, M. biceps femoris, M. rectus abdominis, and M. obliquus externus. The participants performed 10 voluntary pelvic movements (retroversion of the pelvis). The anterior pelvic tilt was measured videographically via marker points on the anterior and posterior superior iliac spine. The EMG data were further processed and normalized to the maximum voluntary contraction. A linear regression analysis was conducted to assess the relationship between changes in the pelvic tilt and muscle activities. Subsequently, a Ward clustering analysis was applied to detect potential muscle activation patterns. The differences between the clusters and the pelvic tilt were examined using ANOVA. Cluster analysis revealed the presence of four clusters with different muscle activation patterns in which the abdominal muscles and dorsal muscle groups were differently involved. However, the gluteus maximus muscle was involved in every activation pattern. It also had the strongest correlation with the changes in pelvic tilt. Different individual muscle patterns are used by different persons to correct pelvic posture, with the gluteus maximus muscle apparently playing the most important role. This can be important for therapy, as different muscle strategies should be trained depending on the individually preferred motor patterns.

Structural modeling of antibody variable regions using deep learning-progress and perspectives on drug discovery.

AlphaFold2 has hallmarked a generational improvement in protein structure prediction. In particular, advances in antibody structure prediction have provided a highly translatable impact on drug discovery. Though AlphaFold2 laid the groundwork for all proteins, antibody-specific applications require adjustments tailored to these molecules, which has resulted in a handful of deep learning antibody structure predictors. Herein, we review the recent advances in antibody structure prediction and relate them to their role in advancing biologics discovery.

A computational method for predicting the aggregation propensity of IgG1 and IgG4(P) mAbs in common storage buffers.

The propensity for some monoclonal antibodies (mAbs) to aggregate at physiological and manufacturing pH values can prevent their use as therapeutic molecules or delay time to market. Consequently, developability assessments are essential to select optimum candidates, or inform on mitigation strategies to avoid potential late-stage failures. These studies are typically performed in a range of buffer solutions because factors such as pH can dramatically alter the aggregation propensity of the test mAbs (up to 100-fold in extreme cases). A computational method capable of robustly predicting the aggregation propensity at the pH values of common storage buffers would have substantial value. Here, we describe a mAb aggregation prediction tool (MAPT) that builds on our previously published isotype-dependent, charge-based model of aggregation. We show that the addition of a homology model-derived hydrophobicity descriptor to our electrostatic aggregation model enabled the generation of a robust mAb developability indicator. To contextualize our aggregation scoring system, we analyzed 97 clinical-stage therapeutic mAbs. To further validate our approach, we focused on six mAbs (infliximab, tocilizumab, rituximab, CNTO607, MEDI1912 and MEDI1912_STT) which have been reported to cover a large range of aggregation propensities. The different aggregation propensities of the case study molecules at neutral and slightly acidic pH were correctly predicted, verifying the utility of our computational method.

Corrigendum: Higher Affinity Antibodies Bind With Lower Hydration and Flexibility in Large Scale Simulations.

[This corrects the article DOI: 10.3389/fimmu.2022.884110.].

Higher Affinity Antibodies Bind With Lower Hydration and Flexibility in Large Scale Simulations.

We have carried out a long-timescale simulation study on crystal structures of nine antibody-antigen pairs, in antigen-bound and antibody-only forms, using molecular dynamics with enhanced sampling and an explicit water model to explore interface conformation and hydration. By combining atomic level simulation and replica exchange to enable full protein flexibility, we find significant numbers of bridging water molecules at the antibody-antigen interface. Additionally, a higher proportion of interactions excluding bulk waters and a lower degree of antigen bound CDR conformational sampling are correlated with higher antibody affinity. The CDR sampling supports enthalpically driven antibody binding, as opposed to entropically driven, in that the difference between antigen bound and unbound conformations do not correlate with affinity. We thus propose that interactions with waters and CDR sampling are aspects of the interface that may moderate antibody-antigen binding, and that explicit hydration and CDR flexibility should be considered to improve antibody affinity prediction and computational design workflows.

Co-evolutionary distance predictions contain flexibility information.

MOTIVATION: Co-evolution analysis can be used to accurately predict residue-residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift from predicting binary contacts to predict distances between pairs of residues. These developments have significantly improved the accuracy of de novo prediction of static protein structures. With AlphaFold2 lifting the accuracy of some predicted protein models close to experimental levels, structure prediction research will move on to other challenges. One of those areas is the prediction of more than one conformation of a protein. Here, we examine the potential of residue-residue distance predictions to be informative of protein flexibility rather than simply static structure. RESULTS: We used DMPfold to predict distance distributions for every residue pair in a set of proteins that showed both rigid and flexible behaviour. Residue pairs that were in contact in at least one reference structure were classified as rigid, flexible or neither. The predicted distance distribution of each residue pair was analysed for local maxima of probability indicating the most likely distance or distances between a pair of residues. We found that rigid residue pairs tended to have only a single local maximum in their predicted distance distributions while flexible residue pairs more often had multiple local maxima. These results suggest that the shape of predicted distance distributions contains information on the rigidity or flexibility of a protein and its constituent residues. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Chemical Synthesis of Knob Domain Antibody Fragments.

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist.

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Structural diversity of B-cell receptor repertoires along the B-cell differentiation axis in humans and mice.

Most current analysis tools for antibody next-generation sequencing data work with primary sequence descriptors, leaving accompanying structural information unharnessed. We have used novel rapid methods to structurally characterize the complementary-determining regions (CDRs) of more than 180 million human and mouse B-cell receptor (BCR) repertoire sequences. These structurally annotated CDRs provide unprecedented insights into both the structural predetermination and dynamics of the adaptive immune response. We show that B-cell types can be distinguished based solely on these structural properties. Antigen-unexperienced BCR repertoires use the highest number and diversity of CDR structures and these patterns of naïve repertoire paratope usage are highly conserved across subjects. In contrast, more differentiated B-cells are more personalized in terms of CDR structure usage. Our results establish the CDR structure differences in BCR repertoires and have applications for many fields including immunodiagnostics, phage display library generation, and "humanness" assessment of BCR repertoires from transgenic animals. The software tool for structural annotation of BCR repertoires, SAAB+, is available at https://github.com/oxpig/saab_plus.

Computational approaches to therapeutic antibody design: established methods and emerging trends.

Antibodies are proteins that recognize the molecular surfaces of potentially noxious molecules to mount an adaptive immune response or, in the case of autoimmune diseases, molecules that are part of healthy cells and tissues. Due to their binding versatility, antibodies are currently the largest class of biotherapeutics, with five monoclonal antibodies ranked in the top 10 blockbuster drugs. Computational advances in protein modelling and design can have a tangible impact on antibody-based therapeutic development. Antibody-specific computational protocols currently benefit from an increasing volume of data provided by next generation sequencing and application to related drug modalities based on traditional antibodies, such as nanobodies. Here we present a structured overview of available databases, methods and emerging trends in computational antibody analysis and contextualize them towards the engineering of candidate antibody therapeutics.

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.

Reconstruction of apo A2A receptor activation pathways reveal ligand-competent intermediates and state-dependent cholesterol hotspots.

G-protein coupled receptors (GPCRs) play a pivotal role in transmitting signals at the cellular level. Structural insights can be exploited to support GPCR structure-based drug discovery endeavours. Despite advances in GPCR crystallography, active state structures are scarce. Molecular dynamics (MD) simulations have been used to explore the conformational landscape of GPCRs. Efforts have been made to retrieve active state conformations starting from inactive structures, however to date this has not been possible without using an energy bias. Here, we reconstruct the activation pathways of the apo adenosine receptor (A2A), starting from an inactive conformation, by applying adaptive sampling MD combined with a goal-oriented scoring function. The reconstructed pathways reconcile well with experiments and help deepen our understanding of A2A regulatory mechanisms. Exploration of the apo conformational landscape of A2A reveals the existence of ligand-competent states, active intermediates and state-dependent cholesterol hotspots of relevance for drug discovery. To the best of our knowledge this is the first time an activation process has been elucidated for a GPCR starting from an inactive structure only, using a non-biased MD approach, opening avenues for the study of ligand binding to elusive yet pharmacologically relevant GPCR states.

B-cell epitopes: Discontinuity and conformational analysis.

Peptide vaccines have many potential advantages over conventional ones including low cost, lack of need for cold-chain storage, safety and specificity. However, it is well known that approximately 90% of B-cell epitopes (BCEs) are discontinuous in nature making it difficult to mimic them for creating vaccines. In this study, the degree of discontinuity in B-cell epitopes and their conformational nature is examined. The discontinuity of B-cell epitopes is analyzed by defining 'regions' (consisting of at least three antibody-contacting residues each separated by ≤3 residues) and small fragments (antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm has been developed that classifies each region's shape as straight, curved or folded on the basis that straight and folded regions are more likely to retain their native conformation as isolated peptides. We have investigated the structures of 488 B-cell epitopes from which 1282 regions and 1018 fragments have been identified. 90% of epitopes have five or fewer regions and five or fewer fragments with 14% containing only one region and 4% being truly linear (i.e. having one region and no fragments). Of the 1282 regions, 508 are straight in shape, 626 are curved and 148 are folded.

The Aminotriazole Antagonist Cmpd-1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor.

The widely expressed G-protein coupled receptors (GPCRs) are versatile signal transducer proteins that are attractive drug targets but structurally challenging to study. GPCRs undergo a number of conformational rearrangements when transitioning from the inactive to the active state but have so far been believed to adopt a fairly conserved inactive conformation. Using 19 F NMR spectroscopy and advanced molecular dynamics simulations we describe a novel inactive state of the adenosine 2A receptor which is stabilised by the aminotriazole antagonist Cmpd-1. We demonstrate that the ligand stabilises a unique conformation of helix V and present data on the putative binding mode of the compound involving contacts to the transmembrane bundle as well as the extracellular loop 2.

SCALOP: sequence-based antibody canonical loop structure annotation.

MOTIVATION: Canonical forms of the antibody complementarity-determining regions (CDRs) were first described in 1987 and have been redefined on multiple occasions since. The canonical forms are often used to approximate the antibody binding site shape as they can be predicted from sequence. A rapid predictor would facilitate the annotation of CDR structures in the large amounts of repertoire data now becoming available from next generation sequencing experiments. RESULTS: SCALOP annotates CDR canonical forms for antibody sequences, supported by an auto-updating database to capture the latest cluster information. Its accuracy is comparable to that of a standard structural predictor but it is 800 times faster. The auto-updating nature of SCALOP ensures that it always attains the best possible coverage. AVAILABILITY AND IMPLEMENTATION: SCALOP is available as a web application and for download under a GPLv3 license at opig.stats.ox.ac.uk/webapps/scalop. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Filtering Next-Generation Sequencing of the Ig Gene Repertoire Data Using Antibody Structural Information.

Next-generation sequencing of the Ig gene repertoire (Ig-seq) produces large volumes of information at the nucleotide sequence level. Such data have improved our understanding of immune systems across numerous species and have already been successfully applied in vaccine development and drug discovery. However, the high-throughput nature of Ig-seq means that it is afflicted by high error rates. This has led to the development of error-correction approaches. Computational error-correction methods use sequence information alone, primarily designating sequences as likely to be correct if they are observed frequently. In this work, we describe an orthogonal method for filtering Ig-seq data, which considers the structural viability of each sequence. A typical natural Ab structure requires the presence of a disulfide bridge within each of its variable chains to maintain the fold. Our Ab Sequence Selector (ABOSS) uses the presence/absence of this bridge as a way of both identifying structurally viable sequences and estimating the sequencing error rate. On simulated Ig-seq datasets, ABOSS is able to identify more than 99% of structurally viable sequences. Applying our method to six independent Ig-seq datasets (one mouse and five human), we show that our error calculations are in line with previous experimental and computational error estimates. We also show how ABOSS is able to identify structurally impossible sequences missed by other error-correction methods.

Observed Antibody Space: A Resource for Data Mining Next-Generation Sequencing of Antibody Repertoires.

Abs are immune system proteins that recognize noxious molecules for elimination. Their sequence diversity and binding versatility have made Abs the primary class of biopharmaceuticals. Recently, it has become possible to query their immense natural diversity using next-generation sequencing of Ig gene repertoires (Ig-seq). However, Ig-seq outputs are currently fragmented across repositories and tend to be presented as raw nucleotide reads, which means nontrivial effort is required to reuse the data for analysis. To address this issue, we have collected Ig-seq outputs from 55 studies, covering more than half a billion Ab sequences across diverse immune states, organisms (primarily human and mouse), and individuals. We have sorted, cleaned, annotated, translated, and numbered these sequences and make the data available via our Observed Antibody Space (OAS) resource at http://antibodymap.org The data within OAS will be regularly updated with newly released Ig-seq datasets. We believe OAS will facilitate data mining of immune repertoires for improved understanding of the immune system and development of better biotherapeutics.

Structurally Mapping Antibody Repertoires.

Every human possesses millions of distinct antibodies. It is now possible to analyze this diversity via next-generation sequencing of immunoglobulin genes (Ig-seq). This technique produces large volume sequence snapshots of B-cell receptors that are indicative of the antibody repertoire. In this paper, we enrich these large-scale sequence datasets with structural information. Enriching a sequence with its structural data allows better approximation of many vital features, such as its binding site and specificity. Here, we describe the structural annotation of antibodies pipeline that maps the outputs of large Ig-seq experiments to known antibody structures. We demonstrate the viability of our protocol on five separate Ig-seq datasets covering ca. 35 m unique amino acid sequences from ca. 600 individuals. Despite the great theoretical diversity of antibodies, we find that the majority of sequences coming from such studies can be reliably mapped to an existing structure.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and ß2-microglobulin (ß2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to ß2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density.

In macromolecular crystallography, the rigorous detection of changed states (for example, ligand binding) is difficult unless signal is strong. Ambiguous ('weak' or 'noisy') density is experimentally common, since molecular states are generally only fractionally present in the crystal. Existing methodologies focus on generating maximally accurate maps whereby minor states become discernible; in practice, such map interpretation is disappointingly subjective, time-consuming and methodologically unsound. Here we report the PanDDA method, which automatically reveals clear electron density for the changed state-even from inaccurate maps-by subtracting a proportion of the confounding 'ground state'; changed states are objectively identified from statistical analysis of density distributions. The method is completely general, implying new best practice for all changed-state studies, including the routine collection of multiple ground-state crystals. More generally, these results demonstrate: the incompleteness of atomic models; that single data sets contain insufficient information to model them fully; and that accuracy requires further map-deconvolution approaches.