Analysis of AlphaMissense data in different protein groups and structural context.

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of missense variants. Since heterogenous performance on different types of proteins can be expected, we assessed the efficacy of AlphaMissense across several protein groups (e.g. soluble, transmembrane, and mitochondrial proteins) and regions (e.g. intramembrane, membrane interacting, and high confidence AlphaFold segments) using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on disordered datasets and ClinVar data related to the CFTR ABC protein. However, a superior performance was shown when benchmarked against the high quality CFTR2 database. Our results with CFTR emphasizes AlphaMissense's potential in pinpointing functional hot spots, with its performance likely surpassing benchmarks calculated from ClinVar and ProteinGym datasets.

Truncated titin is structurally integrated into the human dilated cardiomyopathic sarcomere.

Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the TTNtv+ samples. Full-length titin was reduced in TTNtv+ compared with TTNtv- samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Imaging the Infection Cycle of T7 at the Single Virion Level.

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage-displayed isotropic spatial orientation. The viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle.

Comprehensive Collection and Prediction of ABC Transmembrane Protein Structures in the AI Era of Structural Biology.

The number of unique transmembrane (TM) protein structures doubled in the last four years, which can be attributed to the revolution of cryo-electron microscopy. In addition, AlphaFold2 (AF2) also provided a large number of predicted structures with high quality. However, if a specific protein family is the subject of a study, collecting the structures of the family members is highly challenging in spite of existing general and protein domain-specific databases. Here, we demonstrate this and assess the applicability and usability of automatic collection and presentation of protein structures via the ABC protein superfamily. Our pipeline identifies and classifies transmembrane ABC protein structures using the PFAM search and also aims to determine their conformational states based on special geometric measures, conftors. Since the AlphaFold database contains structure predictions only for single polypeptide chains, we performed AF2-Multimer predictions for human ABC half transporters functioning as dimers. Our AF2 predictions warn of possibly ambiguous interpretation of some biochemical data regarding interaction partners and call for further experiments and experimental structure determination. We made our predicted ABC protein structures available through a web application, and we joined the 3D-Beacons Network to reach the broader scientific community through platforms such as PDBe-KB.

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the ß-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Development, structure and mechanics of a synthetic E. coli outer membrane model.

The outer membrane (OM) of Gram-negative bacteria is a complex asymmetric bilayer containing lipids, lipopolysaccharides (LPS) and proteins. While it is a mechanical and chemical barrier, it is also the primary surface of bacterial recognition processes that involve infection by and of the bacterium. Uncovering the mechanisms of these biological functions has been hampered by the lack of suitable model systems. Here we report the step-by-step assembly of a synthetic OM model from its fundamental components. To enable the efficient formation of a supported lipid bilayer at room temperature, dimyristoyl-phosphocholine (DMPC) was used as the lipid component to which we progressively added LPS and OM proteins. The assembled system enabled us to explore the contribution of the molecular components to the topographical structure and stability of the OM. We found that LPS prefers solid-state membrane regions and forms stable vesicles in the presence of divalent cations. LPS can gradually separate from DMPC membranes to form independent vesicles, pointing at the dynamic nature of the lipid-LPS system. The addition of OM proteins from E. coli and saturating levels of LPS to DMPC liposomes resulted in a thicker and more stable bilayer the surface of which displayed a nanoscale texture formed of parallel, curved, long (>500 nm) stripes spaced apart with a 15 nm periodicity. The synthetic membrane may facilitate the investigation of binding and recognition processes on the surface of Gram-negative bacteria.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations.

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

The MemMoRF database for recognizing disordered protein regions interacting with cellular membranes.

Protein and lipid membrane interactions play fundamental roles in a large number of cellular processes (e.g. signalling, vesicle trafficking, or viral invasion). A growing number of examples indicate that such interactions can also rely on intrinsically disordered protein regions (IDRs), which can form specific reversible interactions not only with proteins but also with lipids. We named IDRs involved in such membrane lipid-induced disorder-to-order transition as MemMoRFs, in an analogy to IDRs exhibiting disorder-to-order transition upon interaction with protein partners termed Molecular Recognition Features (MoRFs). Currently, both the experimental detection and computational characterization of MemMoRFs are challenging, and information about these regions are scattered in the literature. To facilitate the related investigations we generated a comprehensive database of experimentally validated MemMoRFs based on manual curation of literature and structural data. To characterize the dynamics of MemMoRFs, secondary structure propensity and flexibility calculated from nuclear magnetic resonance chemical shifts were incorporated into the database. These data were supplemented by inclusion of sentences from papers, functional data and disease-related information. The MemMoRF database can be accessed via a user-friendly interface at https://memmorf.hegelab.org, potentially providing a central resource for the characterization of disordered regions in transmembrane and membrane-associated proteins.

Single-Molecule Mechanics in Ligand Concentration Gradient.

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

Discovering the chloride pathway in the CFTR channel.

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.

Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

An increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics calculations were performed to characterize the energetics of membrane insertion illuminating functionally crucial regions. In summary, the proposed metrics contribute to deeper understanding of ABC membrane proteins' structural features, structure validation, and analysis of movements observed in a molecular dynamics trajectory. Moreover, the concept of standardized metrics can be applied not only to ABC membrane protein structures (http://conftors.hegelab.org).

Molecular dynamics of the cryo-EM CFTR structure.

Cystic fibrosis (CF), a lethal monogenic disease, is caused by mutant variants of the CF transmembrane conductance regulator (CFTR). Recent advances in single molecule cryo-EM methods enabled structural determination of full-length human and zebrafish CFTR, achieving an important milestone for CF drug development. To relate these structures to the gating cycle, we examined its dynamic features using molecular dynamics simulations. Our results show that the nucleotide binding domains (NBDs) in this bottom-open apo conformation exhibit motions related to dimerization and the bottom-closed apo CFTR model indicates opening of NBDs in contrast to transporters. These observations help in understanding the properties of CFTR chloride channel distinct from transporters and in proper interpretation of available structural information on this ABC protein.

ABCMdb reloaded: updates on mutations in ATP binding cassette proteins.

ABC (ATP-Binding Cassette) proteins with altered function are responsible for numerous human diseases. To aid the selection of positions and amino acids for ABC structure/function studies we have generated a database, ABCMdb (Gyimesi et al. , ABCMdb: a database for the comparative analysis of protein mutations in ABC transporters, and a potential framework for a general application. Hum Mutat 2012; 33:1547-1556.), with interactive tools. The database has been populated with mentions of mutations extracted from full text papers, alignments and structural models. In the new version of the database we aimed to collect the effect of mutations from databases including ClinVar. Because of the low number of available data, even in the case of the widely studied disease-causing ABC proteins, we also included the possible effects of mutations based on SNAP2 and PROVEAN predictions. To aid the interpretation of variations in non-coding regions, the database was supplemented with related DNA level information. Our results emphasize the importance of in silico predictions because of the sparse information available on variants and suggest that mutations at analogous positions in homologous ABC proteins have a strong predictive power for the effects of mutations. Our improved ABCMdb advances the design of both experimental studies and meta-analyses in order to understand drug interactions of ABC proteins and the effects of mutations on functional expression. Database URL: http://abcm2.hegelab.org.

Effects of the gout-causing Q141K polymorphism and a CFTR ΔF508 mimicking mutation on the processing and stability of the ABCG2 protein.

ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the ΔF142 ABCG2, corresponding to the ΔF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. The processing and localization of full length ABCG2 variants were investigated in mammalian cells, followed by Western blotting and confocal microscopy, respectively. Folding and stability were examined by limited proteolysis of Sf9 insect cell membranes expressing these ABCG2 constructs. Stability of isolated nucleotide binding domains, expressed in and purified from bacteria, was studied by CD spectroscopy. We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. In contrast, the ΔF142 mutant has major processing and folding defects, and no ATPase function. We suggest that although these mutations are both localized within the NBD, based on molecular modeling their contribution to the ABCG2 structure and function is different, thus rescue strategies may be devised accordingly.

ABCMdb: a database for the comparative analysis of protein mutations in ABC transporters, and a potential framework for a general application.

To overcome the pathological phenomena caused by altered function of ABC (ATP Binding Cassette) proteins, their mechanisms of action are extensively investigated, often involving the design of mutant constructs for experiments. Designing mutagenetic constructs, interpreting the result of mutagenetic experiments, and finding individual genetic variants require an extensive knowledge of previously published mutations. To aid the recapitulation of mutations described in the literature, we set up a database of ABC protein mutations (ABCMdb) extracted from full-text papers using an automatic mining approach. We have also developed a Web application interface to compare mutations in different ABC proteins using sequence alignments and to interactively map the mutations to 3D structural models. Currently our database contains protein mutations published for ABCB1, ABCB11, ABCC1, ABCC6, ABCC7, and the proteins of the ABCG subfamily. The database will be extended to include other members and subfamilies, and to provide information on whether or not a mutation is disease causing, represents a high-incidence polymorphism, or was generated only in vitro. The ABCMdb database should already help to compare the effects of mutations at homologous positions in different ABC proteins, and its interactive tools aim to advance the design of experiments for a wider range of proteins.

Identification and correction of abnormal, incomplete and mispredicted proteins in public databases.

BACKGROUND: Despite significant improvements in computational annotation of genomes, sequences of abnormal, incomplete or incorrectly predicted genes and proteins remain abundant in public databases. Since the majority of incomplete, abnormal or mispredicted entries are not annotated as such, these errors seriously affect the reliability of these databases. Here we describe the MisPred approach that may provide an efficient means for the quality control of databases. The current version of the MisPred approach uses five distinct routines for identifying abnormal, incomplete or mispredicted entries based on the principle that a sequence is likely to be incorrect if some of its features conflict with our current knowledge about protein-coding genes and proteins: (i) conflict between the predicted subcellular localization of proteins and the absence of the corresponding sequence signals; (ii) presence of extracellular and cytoplasmic domains and the absence of transmembrane segments; (iii) co-occurrence of extracellular and nuclear domains; (iv) violation of domain integrity; (v) chimeras encoded by two or more genes located on different chromosomes. RESULTS: Analyses of predicted EnsEMBL protein sequences of nine deuterostome (Homo sapiens, Mus musculus, Rattus norvegicus, Monodelphis domestica, Gallus gallus, Xenopus tropicalis, Fugu rubripes, Danio rerio and Ciona intestinalis) and two protostome species (Caenorhabditis elegans and Drosophila melanogaster) have revealed that the absence of expected signal peptides and violation of domain integrity account for the majority of mispredictions. Analyses of sequences predicted by NCBI's GNOMON annotation pipeline show that the rates of mispredictions are comparable to those of EnsEMBL. Interestingly, even the manually curated UniProtKB/Swiss-Prot dataset is contaminated with mispredicted or abnormal proteins, although to a much lesser extent than UniProtKB/TrEMBL or the EnsEMBL or GNOMON-predicted entries. CONCLUSION: MisPred works efficiently in identifying errors in predictions generated by the most reliable gene prediction tools such as the EnsEMBL and NCBI's GNOMON pipelines and also guides the correction of errors. We suggest that application of the MisPred approach will significantly improve the quality of gene predictions and the associated databases.

Modules, multidomain proteins and organismic complexity.

Originally the term 'protein module' was coined to distinguish mobile domains that frequently occur as building blocks of diverse multidomain proteins from 'static' domains that usually exist only as stand-alone units of single-domain proteins. Despite the widespread use of the term 'mobile domain', the distinction between static and mobile domains is rather vague as it is not easy to quantify the mobility of domains. In the present work we show that the most appropriate measure of the mobility of domains is the number of types of local environments in which a given domain is present. Ranking of domains with respect to this parameter in different evolutionary lineages highlighted marked differences in the propensity of domains to form multidomain proteins. Our analyses have also shown that there is a correlation between domain size and domain mobility: smaller domains are more likely to be used in the construction of multidomain proteins, whereas larger domains are more likely to be static, stand-alone domains. It is also shown that shuffling of a limited set of modules was facilitated by intronic recombination in the metazoan lineage and this has contributed significantly to the emergence of novel complex multidomain proteins, novel functions and increased organismic complexity of metazoa.

Hydroxamate-based peptide inhibitors of matrix metalloprotease 2.

There is major interest in designing inhibitors for matrix metalloproteinase 2 (MMP-2, gelatinase A) since this enzyme is known to be involved in pathological processes such as tumor invasion or rheumatoid arthritis. The majority of MMP-2 inhibitor candidate drugs block the active site of MMP-2 by binding to its catalytic Zn2+ ion through a chelating (hydroxamate, sulphonate etc.) group. Despite the general interest in designing MMP-2 inhibitors, the results with many of the drug candidates were disappointing, their failure was usually explained by cross-reactions with other MMPs. One way to enhance MMP-2 selectivity is to design inhibitors that interact with both the active site and exosites such as the fibronectin type II (FN2) domains of the enzyme. In the present work, we have examined the inhibitory potential and MMP-2 selectivity of hydroxamates of three groups of peptides known to bind to the collagen-binding FN2 domains of MMP-2. The first type of peptides consisted of collagen-like (Pro-Pro-Gly)(n) repeats, peptides of the second group were identified from a random 15-mer phage display library based on their binding to immobilized FN2 domains of MMP-2. A hydroxamate of peptide p33-42, known to bind to the third FN2 domain of MMP-2 has also been tested. Our studies have shown that these compounds inhibited MMP-2 with IC50 values of 10-100 microM. The fact that their inhibitory potential was nearly identical for MMP-2del, a recombinant version of MMP-2 that lacks the FN2 domains, suggests that inhibition is not mediated by their binding to FN2 domains. It seems likely that the failure to exploit interaction with the FN2 domains is due to the fact that the FN2 domains and the catalytic domain of MMP-2 tumble independently, therefore only a tiny fraction of the conformational isomers can bind peptide hydroxamates via both the active site and the FN2 domain(s).