In silico optimization of protein binding has received a great deal of attention in the recent years. Since in silico prefiltering of strong binders is fast and cheap compared to in vitro library screening methods, the advent of powerful hardware and advanced machine learning algorithms has made this strategy more accessible and preferred. These advances have already impacted the global response to pandemic threats. In this study, we proposed and tested a workflow for designing nanobodies targeting the SARS-CoV-2 spike protein receptor binding domain (S-RBD) using machine learning techniques complemented by molecular dynamics simulations. We evaluated the feasibility of this workflow using a test set of 3 different nanobodies and 2 different S-RBD variants, from in silico design and bacterial expression to binding assays of the designed nanobody mutants. We successfully designed nanobodies that were subsequently tested against both the wild-type (Wuhan type) and the delta variant S-RBD and found 2 of them to be stronger binders compared to the wild-type nanobody. We use this case study to describe both the strengths and weaknesses of this in silico assisted nanobody design strategy.

Non-muscle myosin 2 (NM2) is known to play an important role in myofibroblast transdifferentiation, a hallmark of fibrotic disorders. In a recent JBC article, Southern et al. demonstrate that endogenous S100A4, a calcium- and NM2-binding protein acts as a mechanoeffector in this process. Since extracellular S100A4 is also involved in fibrogenesis by triggering the inflammatory response, this small protein appears to contribute to fibrosis via at least two distinct mechanisms.

Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a K-d of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 mu M. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains—which besides being abundant in proline residues—contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the 1 H N -detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p53 1–100 region by applying a combination of 1 H N - and 1 H α -detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (C α –C β ) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations.

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

The human PDZome represents one of the largest globular domain families in the human proteome, with 266 instances. These globular domains typically interact with C-terminal peptide motifs found in thousands of human proteins. Despite previous efforts, not all PDZ domains have experimentally solved structures and most of their complexes remain to be solved. Here, a simple and cost-effective strategy is proposed for the crystallization of PDZ domains and their complexes. A human annexin A2 fusion tag was used as a crystallization chaperone and the structures of nine PDZ domains were solved, including five domains that had not yet been solved. Finally, these novel experimental structures were compared with AlphaFold predictions and it is speculated how predictions and experimental methods could cooperate in order to investigate the structural landscapes of entire domain families and interactomes.

The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called “tandem SH3”) and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

S100 proteins are small, dimeric, Ca2+-binding proteins of considerable interest due to their associations with cancer and rheumatic and neurodegenerative diseases. They control the functions of numerous proteins by forming protein-protein complexes with them. Several of these complexes were found to display "fuzzy" properties. Examining these highly flexible interactions, however, is a difficult task, especially from a structural biology point of view. Here, we summarize the available in vitro techniques that can be deployed to obtain structural information about these dynamic complexes. We also review the current state of knowledge about the structures of S100 complexes, focusing on their often-asymmetric nature.

The dynamic regulation of protein-protein interactions (PPIs) involves phosphorylation of short liner motifs in disordered protein regions modulating binding affinities. The ribosomal-S6-kinase 1 is capable of binding to scaffold proteins containing PDZ domains through a PDZ-binding motif (PBM) located at the disordered C-terminus of the kinase. Phosphorylation of the PBM dramatically changes the interactome of RSK1 with PDZ domains exerting a fine-tuning mechanism to regulate PPIs. Here we present in detail highly effective biophysical (fluorescence polarization, isothermal calorimetry) and cellular (proteinfragment complementation) methods to study the effect of phosphorylation on RSK1-PDZ interactions that can be also applied to investigate phosphoregulation of other PPIs in signaling pathways.

Abstract S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1 H α -detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4–15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans -proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans -proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

To fully understand the environmental factors that influence crystallization is an enormous task, therefore crystallographers are still forced to work "blindly" trying as many crystallizing conditions and mutations to improve crystal packing as possible. Numerous times these random attempts simply fail even when using state-of-the-art techniques. As an alternative, crystallization chaperones, having good crystal-forming properties, can be invoked. Today, the almost exclusively used such protein is the maltose-binding protein (MBP) and crystallographers need other widely applicable options. Here, we introduce annexin A2 (ANXA2), which has just as good, if not better, crystal-forming ability than the wild-type MBP. Using ANXA2 as heterologous fusion partner, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with the metastasis-associated protein S100A4. p53 TAD forms an asymmetric fuzzy complex with the symmetric S1004 and could interfere with its function.

The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein-protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally-occurring S100 partners in the human proteome. Such information will be precious for future drug design of modulators of S100 pathological activities.

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD(1-60)and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD(1-60)remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting "fuzzy" complex, the clamp-like binding of p53TAD(1-60)relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Delta delta, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.