Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 µM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions.

RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene's importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 µM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.

Intrinsically Disordered Flanking Regions Increase the Affinity of a Transcriptional Coactivator Interaction across Vertebrates.

Interactions between two proteins are often mediated by a disordered region in one protein binding to a groove in a folded interaction domain in the other one. While the main determinants of a certain interaction are typically found within a well-defined binding interface involving the groove, recent studies show that nonspecific contacts by flanking regions may increase the affinity. One example is the coupled binding and folding underlying the interaction between the two transcriptional coactivators NCOA3 (ACTR) and CBP, where the flanking regions of an intrinsically disordered region in human NCOA3 increases the affinity for CBP. However, it is not clear whether this flanking region-mediated effect is a peculiarity of this single protein interaction or if it is of functional relevance in a broader context. To further assess the role of flanking regions in the interaction between NCOA3 and CBP, we analyzed the interaction across orthologs and paralogs (NCOA1, 2, and 3) in human, zebra fish, and ghost shark. We found that flanking regions increased the affinity 2- to 9-fold in the six interactions tested. Conservation of the amino acid sequence is a strong indicator of function. Analogously, the observed conservation of increased affinity provided by flanking regions, accompanied by moderate sequence conservation, suggests that flanking regions may be under selection to promote the affinity between NCOA transcriptional coregulators and CBP.

Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif-mediated interactions.

The interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well-studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2-family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12-residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (KD around 0.1 µM). The affinity of the bay mussel p53TAD/MDM2 complex was lower (KD = 15 µM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (KD > 100 µM). Binding experiments with reconstructed ancestral p53TAD/MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/MDM2 affinity during speciation demonstrate high plasticity of motif-mediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD.

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs.

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions.

Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.

Allostery Frustrates the Experimentalist.

Proteins interact with other proteins, with nucleic acids, lipids, carbohydrates and various small molecules in the living cell. These interactions have been quantified and structurally characterized in numerous studies such that we today have a comprehensive picture of protein structure and function. However, proteins are dynamic and even folded proteins are likely more heterogeneous than they appear in most descriptions. One property of proteins that relies on dynamics and heterogeneity is allostery, the ability of a protein to change structure and function upon ligand binding to an allosteric site. Over the last decades the concept of allostery was broadened to embrace all types of long-range interactions across a protein including purely entropic changes without a conformational change in single protein domains. But with this re-definition came a problem: How do we measure allostery? In this opinion, we discuss some caveats arising from the quantitative description of single-domain allostery from an experimental perspective and how the limitations cannot be separated from the definition of allostery per se. Furthermore, we attempt to tie together allostery with the concept of frustration in an effort to investigate the links between these two complex, and yet general, properties of proteins. We arrive at the conclusion that the sensitivity to perturbation of allosteric networks in single protein domains is too large for the networks to be of significant biological relevance.

A Syntenin Inhibitor Blocks Endosomal Entry of SARS-CoV-2 and a Panel of RNA Viruses.

Viruses are dependent on host factors in order to efficiently establish an infection and replicate. Targeting the interactions of such host factors provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking and has previously been shown to be important for human papilloma virus (HPV) infection. Here, we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection and strongly reduced flavivirus infection, which is completely dependent on receptor-mediated endocytosis for their entry. In conclusion, we have identified a novel broad spectrum antiviral inhibitor that efficiently targets a broad range of RNA viruses.

Determinants of affinity, specificity, and phase separation in a supramodule from Post-synaptic density protein 95.

The post-synaptic density (PSD) is a phase-separated membraneless compartment of proteins including PSD-95 that undergoes morphological alteration in response to synaptic activity. Here, we investigated the interactome of a three-domain supramodule, PDZ3-SH3-GK (PSG) from PSD-95 using bioinformatics to identify potential binding partners, and biophysical methods to characterize the interaction with peptides from these proteins. PSG and the single PDZ3 domain bound similar peptides, but with different specificity. Furthermore, we found that the protein ADGRB1 formed liquid droplets with the PSG supramodule, extending the model for PSD formation. Moreover, certain mutations, introduced outside of the binding pocket in PDZ3, increased the affinity and specificity of the interaction and the size of liquid droplets. Other mutations within the ligand binding pocket lead to a new binding motif specificity. Our results show how the context in terms of supertertiary structure modulates affinity, specificity, and phase separation, and how these properties can evolve by point mutation.

Monoclonal antibodies binding data for SARS-CoV-2 proteins.

SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.

Disordered Regions Flanking the Binding Interface Modulate Affinity between CBP and NCOA.

Recognition motifs that mediate protein-protein interactions are usually embedded within longer intrinsically disordered regions. While binding interfaces involving the recognition motif in such interactions are well studied, less is known about the role of disordered regions flanking the motifs. The interaction between the transcriptional co-activators NCOA3 (ACTR) and CBP is mediated by coupled binding and folding of the two domains CID and NCBD. Here, we used circular dichroism and kinetics to directly quantify the contribution of the adjacent flanking regions of CID to its interaction with NCBD. Using N- and C-terminal combinatorial variants we found that the flanking regions promote binding in an additive fashion while retaining a large degree of disorder in the complex. Experiments at different ionic strengths demonstrated that the increase in affinity is not mediated by electrostatic interactions from the flanking regions. Instead, site-directed mutagenesis and molecular dynamics simulations suggest that binding is promoted by short-lived non-specific hydrophobic contacts between the flanking regions and NCBD. Our findings are consistent with highly frustrated interactions outside of the canonical binding interface resulting in a slightly energetically favorable fuzzy binding. Modulation of affinity via flanking regions could represent a general mechanism for functional regulation by intrinsically disordered protein regions.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described as 'molten-globule like'. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetics we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.

Proteome-scale mapping of binding sites in the unstructured regions of the human proteome.

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display 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. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities.

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 or E7 induces senescence in cervical cancer cells.

Human papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, anogenital and oropharyngeal sites. Continuous expression of the HPV oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 or E7 genes can potentially treat HPV-related cancers. Here we report that CRISPR/Cas9-based knockout of E6 or E7 can trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, E6 or E7-inactivated HeLa cells exhibited characteristic senescence markers like enlarged cell surface area, increased ß-galactosidase expression and loss of lamin B1. Since E6 and E7 are bicistronic transcripts, inactivation of HPV18 E6 resulted in knockout of both E6 and E7 and increasing levels of p53/p21 and pRb/p21, respectively. Knockout of HPV18 E7 resulted in decreased E6 expression with activation of pRb/p21 pathway. Taken together, our study demonstrates cellular senescence as an alternative outcome of HPV oncogene inactivation by CRISPR/Cas9.

Dissecting Inter-domain Cooperativity in the Folding of a Multi Domain Protein.

Correct protein folding underlies all cellular functions. While there are detailed descriptions and a good understanding of protein folding pathways for single globular domains there is a paucity of quantitative data regarding folding of multidomain proteins. We have here investigated the folding of a three-domain supramodule from the protein PSD-95, consisting of one PDZ domain, one SH3 domain and one guanylate kinase-like (GK) domain. This supramodule has previously been shown to work as one functional unit with regard to ligand binding. We used equilibrium and kinetic folding experiments to demonstrate that the PDZ domain folds faster and independently from the SH3-GK tandem, which folds as one cooperative unit. However, concurrent folding of the PDZ domain slows down folding of SH3-GK by non-native interactions, resulting in an off-pathway folding intermediate. Our data contribute to an emerging description of multidomain protein folding in which individual domains cannot a priori be viewed as separate folding units.