Unstructured Region Research Articles

Mapping the Domain Structure and Aggregation Propensity of Proteins Using a Gateway Plasmid Vector System.

Some proteins represent members of conserved families, meaning that their domain structure can be easily predicted by comparison to homologous proteins whose structures have been solved experimentally. Many other proteins, however, do not share significant detectable homology with other proteins, often as results of high amounts of coiled-coil structure and/or intrinsically unstructured regions. These proteins include many whose aggregation is linked to human disease.Here we present a refined and reliable workflow for identifying the domains of such proteins, through cloning of multiple alternative fragments, and testing whether they form soluble, folded structures when expressed as recombinant peptides in E. coli, through the use of size exclusion chromatography. By using Gateway recombination for cloning, these fragments can then be rapidly transferred to alternate vectors for testing in mammalian cells. We then specifically illustrate its use for proteins that form pathological aggregates in disease, mapping not just their basic domain structures but also the specific subdomains responsible for aggregation.

Intrinsically disordered protein regions are required for cell wall homeostasis in Bacillus subtilis.

Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.

Targeting androgen receptor phase separation to overcome antiandrogen resistance.

Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid-liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid-liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.

Analysis of Host–Bacteria Protein Interactions Reveals Conserved Domains and Motifs That Mediate Fundamental Infection Pathways

Adhesion and colonization of host cells by pathogenic bacteria depend on protein–protein interactions (PPIs). These interactions are interesting from the pharmacological point of view since new molecules that inhibit host-pathogen PPIs would act as new antimicrobials. Most of these interactions are discovered using high-throughput methods that may display a high false positive rate. The absence of curation of these databases can make the available data unreliable. To address this issue, a comprehensive filtering process was developed to obtain a reliable list of domains and motifs that participate in PPIs between bacteria and human cells. From a structural point of view, our analysis revealed that human proteins involved in the interactions are rich in alpha helix and disordered regions and poorer in beta structure. Disordered regions in human proteins harbor short sequence motifs that are specifically recognized by certain domains in pathogenic proteins. The most relevant domain–domain interactions were validated by AlphaFold, showing that a proper analysis of host-pathogen PPI databases can reveal structural conserved patterns. Domain–motif interactions, on the contrary, were more difficult to validate, since unstructured regions were involved, where AlphaFold could not make a good prediction. Moreover, these interactions are also likely accommodated by post-translational modifications, especially phosphorylation, which can potentially occur in 25–50% of host proteins. Hence, while common structural patterns are involved in host–pathogen PPIs and can be retrieved from available databases, more information is required to properly infer the full interactome. By resolving these issues, and in combination with new prediction tools like Alphafold, new classes of antimicrobials could be discovered from a more detailed understanding of these interactions.

Phase separation of p53 induced by its unstructured basic region and prevented by oncogenic mutations in tetramerization domain

Liquid-liquid phase separation (LLPS) drives the formation of extensive membrane-less compartments to regulate various cellular biological activities both physiologically and pathologically. It has been widely accepted that LLPS is closely related to amyloid diseases and increasing reports have linked this phenomenon to cancers. Mutations of tumor suppressor protein p53 exist in more than half of malignant tumors, making the protein vitally important in cancer research. Recently, p53 was reported to undergo phase separation, which may regulate the function of p53. The molecular mechanism of p53 phase separation and how this process relates to cancer remains largely unclear. Herein, we find that the disordered unstructured basic region (UBR) plays a crucial role in p53 LLPS, driven by electrostatic and hydrophobic interactions. Mutations in the tetramerization domain (TD) disrupt p53 phase separation by preventing the tetramer formation. Furthermore, our results have revealed that, in response to DNA damage in cell, the wild type (WT) p53 undergoes LLPS, while LLPS in oncogenic mutations is diminished or eliminated. The expression of the target gene of p53 decreased significantly with the mutations and cell survival increased with the mutations. Thus, we propose a novel mechanism of p53 carcinogenesis, whereby oncogenic mutations in TD impair the formation of p53 condensates, decreasing the activation of target genes and promoting cancer progression. This study helps to understand the behavior and function of p53 in a different aspect and may provide insights into cancer therapies targeting p53.

In silico analyses of Wnt1 nsSNPs reveal structurally destabilizing variants, altered interactions with Frizzled receptors and its deregulation in tumorigenesis

Wnt1 is the first mammalian Wnt gene, which is discovered as proto-oncogene and in human the gene is located on the chromosome 12q13. Mutations in Wnt1 are reported to be associated with various cancers and other human diseases. The structural and functional consequences of most of the non-synonymous SNPs (nsSNPs), present in the human Wnt1 gene, are not known. In the present work, extensive bioinformatics analyses are used to screen 292 nsSNPs of Wnt1 for predicting pathogenic and harmless polymorphisms. We have identified 10 highly deleterious nsSNPs among which 7 are located within the highly conserved areas. These 10 nsSNPs are also predicted to affect the post-translational modifications of Wnt1. Further, structure based stability analyses of these 10 highly deleterious nsSNPs revealed 8 variants as highly destabilizing. These 8 highly destabilizing variants were shown to have high BC score and high RMSIP score from normal mode analyses. Based on the deformation energies, obtained from the normal mode analyses, variants like G169A, G169S, G331R and G331S were found to be unstable. Molecular Dynamics (MD) simulations revealed structural stability and fluctuation of WT Wnt1 and its prioritized variants. RMSD remained fluctuating mostly between 4 and 5 Å and occasionally between 3.5 and 5.5 Å ranges. RMSF in the CTD region (residues 330–360) of the binding pocket were lower compared to that of WT. Studying the impacts of nsSNPs on the binding interface of Wnt1 and seven Frizzled receptors have predicted substitutions which can stabilize or destabilize the binding interface. We have found that Wnt1 and FZD8-CRD is the best docked complex in our study. MD simulation based analyses of wild type Wnt1-FZD8-CRD complex and the 8 prioritized variants revealed that RMSF was higher in the unstructured regions and RMSD remained fluctuating in the region of 5 Å ± 1 Å. We have also observed differential Wnt1 gene expression pattern in normal, tumor and metastatic conditions across different tissues. Wnt1 gene expression was significantly higher in metastatic tissues of lungs, colon and skin; and was significantly lower in metastatic tissues of breast, esophagus and kidney. We have also found that Wnt1 deregulation is associated with survival outcome in patients with gastric and breast cancer. Furthermore, these computationally screened highly deleterious nsSNPs of Wnt1 can be analyzed in population based genetic studies and may help understand the Wnt1 associated diseases.

An optimally convergent smooth blended B-spline construction for semi-structured quadrilateral and hexahedral meshes

Easy to construct and optimally convergent generalisations of B-splines to unstructured meshes are essential for the application of isogeometric analysis to domains with non-trivial topologies. Nonetheless, especially for hexahedral meshes, the construction of smooth and optimally convergent isogeometric analysis basis functions is still an open question. We introduce a simple partition of unity construction that yields smooth blended B-splines, referred to as SB-splines, on semi-structured quadrilateral and hexahedral meshes, i.e. on mostly structured meshes with sufficiently separated unstructured regions. To this end, we first define the mixed smoothness B-splines that are C0 continuous in the unstructured regions of the mesh but have higher smoothness everywhere else. Subsequently, the SB-splines are obtained by smoothly blending in the physical space the mixed smoothness B-splines with Bernstein bases of equal degree. One of the key novelties of our approach is that the required smooth weight functions are assembled from the available smooth B-splines on the unstructured mesh. The SB-splines are globally smooth, non-negative, have no breakpoints within the elements and reduce to conventional B-splines away from the unstructured regions of the mesh. Although we consider only quadratic mixed smoothness B-splines in this paper, the construction generalises to arbitrary degrees. We demonstrate the excellent performance of SB-splines studying Poisson and biharmonic problems on semi-structured quadrilateral and hexahedral meshes, and numerically establishing their optimal convergence in one and two dimensions.

Efficient reduced order model based on the proper orthogonal decomposition for time-dependent MOC calculations

ABSTRACT An efficient reduced order model (ROM) for time-dependent transport calculations using the method of characteristics (MOC) is proposed. In the present ROM, the flux distributions and the net neutron currents between the adjacent unstructured mesh regions are taken from the MOC solution. Then, the coefficient matrices for the MOC-equivalent diffusion equation are reconstructed from them. The proper orthogonal decomposition (POD) is applied for the MOC-equivalent diffusion equation to reduce the degree of freedoms (DOFs) using the orthogonal bases obtained by the singular value decomposition (SVD) for the sampled MOC solution. The accuracy and computation time of the present ROM are verified in the C5G7-TD 2D benchmark problem. The calculation results show that the present ROM enables us approximately 5000–6000 times faster computation than the full order model (FOM) for kinetic calculations itself in the present calculation condition. The present method can be substituted as real-time simulations without the spatial homogenization when typical flux distributions and the net neutron currents of a target problem can be precalculated.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Machine-learning analysis of intrinsically disordered proteins identifies key factors that contribute to neurodegeneration-related aggregation.

Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates.

Shelterin is a dimeric complex with extensive structural heterogeneity

Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

Exploring Manually Curated Annotations of Intrinsically Disordered Proteins with DisProt

DisProt is the major repository of manually curated data for intrinsically disordered proteins collected from the literature. Although lacking a stable three-dimensional structure under physiological conditions, intrinsically disordered proteins carry out a plethora of biological functions, some of them directly arising from their flexible nature. A growing number of scientific studies have been published during the last few decades to shed light on their unstructured state, their binding modes, and their functions. DisProt makes use of a team of expert biocurators to provide up-to-date annotations of intrinsically disordered proteins from the literature, making them available to the scientific community. Here we present a comprehensive description on how to use DisProt in different contexts and provide a detailed explanation of how to explore and interpret manually curated annotations of intrinsically disordered proteins. We describe how to search DisProt annotations, both using the web interface and the API for programmatic access. Finally, we explain how to visualize and interpret a DisProt entry, the SARS-CoV-2 Nucleoprotein, characterized by the presence of unstructured N-terminal and C-terminal regions and a flexible linker. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Performing a search in DisProt Support Protocol 1: Downloading options Support Protocol 2: Programmatic access with DisProt REST API Basic Protocol 2: Exploring the DisProt Ontology page Basic Protocol 3: Visualizing and interpreting DisProt entries-the SARS-CoV-2 Nucleoprotein use case.

Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

As a critical node for insulin/IGF signaling, insulin receptor substrate 1 (IRS-1) is essential for metabolic regulation. A long and unstructured C-terminal region of IRS-1 recruits downstream effectors for promoting insulin/IGF signals. However, the underlying molecular basis for this remains elusive. Here, we found that the C-terminus of IRS-1 undergoes liquid-liquid phase separation (LLPS). Both electrostatic and hydrophobic interactions were seen to drive IRS-1 LLPS. Self-association of IRS-1, which was mainly mediated by the 301–600 region, drives IRS-1 LLPS to form insulin/IGF-1 signalosomes. Moreover, tyrosine residues of YXXM motifs, which recruit downstream effectors, also contributed to IRS-1 self-association and LLPS. Impairment of IRS-1 LLPS attenuated its positive effects on insulin/IGF-1 signaling. The metabolic disease-associated G972R mutation impaired the self-association and LLPS of IRS-1. Our findings delineate a mechanism in which LLPS of IRS-1-mediated signalosomes serves as an organizing center for insulin/IGF-1 signaling and implicate the role of aberrant IRS-1 LLPS in metabolic diseases.

Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation.

In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.

Specific Zn(II)-binding site in the C-terminus of Aspf2, a zincophore from Aspergillus fumigatus.

Aspergillus fumigatus, one of the most widespread opportunistic human fungal pathogens, adapts to zinc limitation by secreting a 310 amino acid Aspf2 zincophore, able to specifically bind Zn(II) and deliver it to a transmembrane zinc transporter, ZrfC. In this work, we focus on the thermodynamics of Zn(II) complexes with unstructured regions of Aspf2; basing on a variety of spectrometric and potentiometric data, we show that the C-terminal part has the highest Zn(II)-binding affinity among the potential binding sites, and Ni(II) does not compete with Zn(II) binding to this region. The 14 amino acid Aspf2 C-terminus coordinates Zn(II) via two Cys thiolates and two His imidazoles and it could be considered as a promising A. fumigatus targeting molecule.

Alpha-Synuclein Aggregation Pathway in Parkinson's Disease: Current Status and Novel Therapeutic Approaches.

Following Alzheimer’s, Parkinson’s disease (PD) is the second-most common neurodegenerative disorder, sharing an unclear pathophysiology, a multifactorial profile, and massive social costs worldwide. Despite this, no disease-modifying therapy is available. PD is tightly associated with α-synuclein (α-Syn) deposits, which become organised into insoluble, amyloid fibrils. As a typical intrinsically disordered protein, α-Syn adopts a monomeric, random coil conformation in an aqueous solution, while its interaction with lipid membranes drives the transition of the molecule part into an α-helical structure. The central unstructured region of α-Syn is involved in fibril formation by converting to well-defined, β-sheet rich secondary structures. Presently, most therapeutic strategies against PD are focused on designing small molecules, peptides, and peptidomimetics that can directly target α-Syn and its aggregation pathway. Other approaches include gene silencing, cell transplantation, stimulation of intracellular clearance with autophagy promoters, and degradation pathways based on immunotherapy of amyloid fibrils. In the present review, we sum marise the current advances related to α-Syn aggregation/neurotoxicity. These findings present a valuable arsenal for the further development of efficient, nontoxic, and non-invasive therapeutic protocols for disease-modifying therapy that tackles disease onset and progression in the future.

Hydrogen‐Deuterium Exchange Identifies the Structural Basis of Phase Separation of the Chromosome Passenger Complex

The centromere is the locus that directs accurate chromosome segregation, and a master regulatory complex, the chromosome passenger complex (CPC) is enriched in a region of the mitotic centromere termed the inner centromere. In mitosis, the CPC monitors the connections of the centromere to the mitotic spindle, acting to destabilize incorrect attachments but permitting correct attachments to persist. The CPC contains the Aurora‐B kinase and three regulatory subunits: INCENP, Survivin and Borealin. Recent findings support a role for CPC phase separation in its localization and function in mitotic error correction (Trivedi et al., 2019, Nat. Cell Biol. 21:1127‐1137). Liquid‐liquid phase separation is emerging as a central regulatory principle of cell biology. This form of phase separation is often driven by multivalency of adhesive domains and/or linear motifs. The sequences of these domains are often found in unstructured regions where they are enriched in uncharged polar side chains, charged amino acids, or aromatic residues; additionally, structural plasticity allows them to generate multiple, weakly‐adhesive, inter‐ and intramolecular interactions. Currently there are no techniques to globally map the motifs on proteins that drive the multivalent interactions. In this context, we now employ hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) to study dynamics of CPC phase separation. We have identified four regions within the regulatory CPC subunits that upon phase separation exhibit differential HX (three slower and one faster). Importantly, three of these regions map to structured regions of proteins while one maps to the expected intrinsically disordered regions. Each of the four regions is enriched with charged amino acids. Mutation of key residues in one of the differential HX regions within the INCENP subunit diminish droplet size and quantity, suggesting that we have successfully mapped a key surface for the phase separation properties of the CPC. In broader context we have shown for the first time how HXMS coupled with structural data and bioinformatic analysis can be applied to study molecular basics of protein liquid‐liquid phase separation.

Proteome‐scale amino‐acid resolution footprinting of protein‐binding sites in the intrinsically disordered regions

Protein‐protein interactions are central to cell function. Numerous studies have provided large‐scale information on human protein‐protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type‐specific interactions disproportionately under‐represented. I will present our efforts towards finding the short linear motif (SLiM)‐based interactions of a variety of protein domains. I will present an optimized proteomic peptide‐phage display (ProP‐PD) library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay, and the tools and guidelines we have defined for processing the data (1). Using the approach we identified >2,000 interaction pairs for 35 known SLiM‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. The amino acid resolution binding site information can be used to pin‐point functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. Finally, I will describe how we developed the approach for large‐scale discovery of coronavirus‐host factor protein interactions (2). We screened more than 130 protein domains for binding to viral peptides, and translated the high‐resolution information on direct virus‐host interactions to a specific peptide‐based antiviral inhibitor of an interaction between the G3BP1/2 proteins and an ΦxFG peptide motif in the SARS‐CoV‐2 nucleocapsid (N) protein. ProP‐PD may thus be used both to illuminate the motif‐based part of the interactome and to uncover leads for innovative inhibitor design.References Benz et al., (2022) Proteome‐scale mapping of binding sites in the unstructured regions of the human proteome. Mol Syst Biol. 2022 Jan;18(1):e10584. Kruse et al., (2021) Large scale discovery of coronavirus‐host factor protein interaction motifs reveals SARS‐CoV‐2 specific mechanisms and vulnerabilities. Nat Commun. 2021 Nov 19;12(1):6761.

Membranome 3.0: Database of single-pass membrane proteins with AlphaFold models.

The Membranome database provides comprehensive structural information on single‐pass (i.e., bitopic) membrane proteins from six evolutionarily distant organisms, including protein–protein interactions, complexes, mutations, experimental structures, and models of transmembrane α‐helical dimers. We present a new version of this database, Membranome 3.0, which was significantly updated by revising the set of 5,758 bitopic proteins and incorporating models generated by AlphaFold 2 in the database. The AlphaFold models were parsed into structural domains located at the different membrane sides, modified to exclude low‐confidence unstructured terminal regions and signal sequences, validated through comparison with available experimental structures, and positioned with respect to membrane boundaries. Membranome 3.0 was re‐developed to facilitate visualization and comparative analysis of multiple 3D structures of proteins that belong to a specified family, complex, biological pathway, or membrane type. New tools for advanced search and analysis of proteins, their interactions, complexes, and mutations were included. The database is freely accessible at https://membranome.org.

Dimerization of the pulmonary surfactant protein C in a membrane environment.

Surfactant protein C (SP-C) has several functions in pulmonary surfactant. These include the transfer of lipids between different membrane structures, a role in surfactant recycling and homeostasis, and involvement in modulation of the innate defense system. Despite these important functions, the structures of functional SP-C complexes have remained unclear. SP-C is known to exist as a primarily α-helical structure with an apparently unstructured N-terminal region, yet there is recent evidence that the functions of SP-C could be associated with the formation of SP-C dimers and higher oligomers. In this work, we used molecular dynamics simulations, two-dimensional umbrella sampling, and well-tempered metadynamics to study the details of SP-C dimerization. The results suggest that SP-C dimerizes in pulmonary surfactant membranes, forming dimers of different topologies. The simulations identified a dimerization motif region V21xxxVxxxGxxxM33 that is much larger than the putative A30xxxG34 motif that is commonly assumed to control the dimerization of some α-helical transmembrane domains. The results provide a stronger basis for elucidating how SP-C functions in concert with other surfactant proteins.