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The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The infection is caused when Spike-protein (S-protein) present on the surface of SARS-CoV-2 interacts with human cell surface receptor, Angiotensin-converting enzyme 2 (ACE2). This binding facilitates SARS-CoV-2 genome entry into the human cells, which in turn causes infection. Since the beginning of the pandemic, many different therapies have been developed to combat COVID-19, including treatment and prevention. This review is focused on the currently adapted and certain other potential therapies for COVID-19 treatment, which include drug repurposing, vaccines and drug-free therapies. The efficacy of various treatment options is constantly being tested through clinical trials and in vivo studies before they are made medically available to the public. more...

Hsp90 is a molecular chaperone responsible for regulating proteostasis under physiological and pathological conditions. Its central role in a range of diseases and potential as a drug target has focused efforts to understand its mechanisms and biological functions and to identify modulators that may form the basis for therapies. The 10th international conference on the Hsp90 chaperone machine was held in Switzerland in October 2022. The meeting was organized by Didier Picard (Geneva, Switzerland) and Johannes Buchner (Garching, Germany) with an advisory committee of Olivier Genest, Mehdi Mollapour, Ritwick Sawarkar, and Patricija van Oosten-Hawle. This was a much anticipated first in-person meeting of the Hsp90 community since 2018 after the COVID-19 pandemic led to the postponement of the 2020 meeting. The conference remained true to the tradition of sharing novel data ahead of publication, providing unparalleled depth of insight for both experts and newcomers to the field. more...

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins. more...

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its β-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its β-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface. more...

Protein-ligand binding free-energy calculations using molecular dynamics (MD) simulations have emerged as a powerful tool for in silico drug design. Here, we present results obtained with the ARROW force field (FF)─a multipolar polarizable and physics-based model with all parameters fitted entirely to high-level ab initio quantum mechanical (QM) calculations. ARROW has already proven its ability to determine solvation free energy of arbitrary neutral compounds with unprecedented accuracy. The ARROW FF parameterization is now extended to include coverage of all amino acids including charged groups, allowing molecular simulations of a series of protein-ligand systems and prediction of their relative binding free energies. We ensure adequate sampling by applying a novel technique that is based on coupling the Hamiltonian Replica exchange (HREX) with a conformation reservoir generated via potential softening and nonequilibrium MD. ARROW provides predictions with near chemical accuracy (mean absolute error of ∼0.5 kcal/mol) for two of the three protein systems studied here (MCL1 and Thrombin). The third protein system (CDK2) reveals the difficulty in accurately describing dimer interaction energies involving polar and charged species. Overall, for all of the three protein systems studied here, ARROW FF predicts relative binding free energies of ligands with a similar accuracy level as leading nonpolarizable force fields. more...

Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags. more...

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1–4. However, the receptor for NeoCoV—the closest known MERS-CoV relative found in bats—remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD–ACE2 binding interface involving protein–glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337–342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat. more...

In native systems, gene expression is regulated by RNA binding proteins. Such proteins have been the target of a great deal of recent research interest, due to the potential for harnessing these regulatory effects for the construction of new biotechnological tools. In particular, focus has been targeted on building synthetic RNA binding proteins for sequence-specific targeting of new RNA transcripts. Pentatricopeptide repeat (PPR) proteins make compelling candidates as synthetic RNA binding proteins to target and bind RNA transcripts of interest, due to their defined RNA binding "code", modular structure, and native capability to deliver catalytic C-terminal domains. In this review, we present a summary of up-to-date understanding of RNA site recognition by PPR proteins, progress towards the design of synthetic PPR proteins for RNA targeting in vitro and in vivo, highlight key areas for further research around these proteins and present an outlook for future applications for synthetic PPR proteins as biotechnological tools. more...

Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and β-actin were present as nonspecifically bound protein to the beads. p53 and β-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and β-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins. more...

As a vital target for the development of novel anti-cancer drugs, human concentrative nucleoside transporter 3 (hCNT3) has been widely concerned. Nevertheless, the lack of a comprehensive understanding of molecular interactions and motion mechanism has greatly hindered the development of novel inhibitors against hCNT3. In this paper, molecular recognition of hCNT3 with uridine was investigated with molecular docking, conventional molecular dynamics (CMD) simulations and adaptive steered molecular dynamics (ASMD) simulations; and then, the uridine derivatives with possibly highly inhibitory activity were designed. The result of CMD showed that more water-mediated H-bonds and lower binding free energy both explained higher recognition ability and transported efficiency of hCNT3. While during the ASMD simulation, nucleoside transport process involved the significant side-chain flip of residues F321 and Q142, a typical substrate-induced conformational change. By considering electronegativity, atomic radius, functional group and key H-bonds factors, 25 novel uridine derivatives were constructed. Subsequently, the receptor-ligand binding free energy was predicted by solvated interaction energy (SIE) method to determine the inhibitor c8 with the best potential performance. This work not only revealed molecular recognition and release mechanism of uridine with hCNT3, but also designed a series of uridine derivatives to obtain lead compounds with potential high activity. more...

Fc γreceptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification. more...

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using more...

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding. more...

The development of accurate transferable force fields is key to realizing the full potential of atomistic modeling in the study of biological processes such as protein-ligand binding for drug discovery. State-of-the-art transferable force fields, such as those produced by the Open Force Field Initiative, use modern software engineering and automation techniques to yield accuracy improvements. However, force field torsion parameters, which must account for many stereoelectronic and steric effects, are considered to be less transferable than other force field parameters and are therefore often targets for bespoke parametrization. Here, we present the Open Force Field QCSubmit and BespokeFit software packages that, when combined, facilitate the fitting of torsion parameters to quantum mechanical reference data at scale. We demonstrate the use of QCSubmit for simplifying the process of creating and archiving large numbers of quantum chemical calculations, by generating a dataset of 671 torsion scans for druglike fragments. We use BespokeFit to derive individual torsion parameters for each of these molecules, thereby reducing the root-mean-square error in the potential energy surface from 1.1 kcal/mol, using the original transferable force field, to 0.4 kcal/mol using the bespoke version. Furthermore, we employ the bespoke force fields to compute the relative binding free energies of a congeneric series of inhibitors of the TYK2 protein, and demonstrate further improvements in accuracy, compared to the base force field (MUE reduced from 0.56 more...

Understanding ligand binding kinetics and thermodynamics, which involves investigating the free, transient, and final complex conformations, is important in fundamental studies and applications for chemical and biomedical systems. Examining the important but transient ligand-protein-bound conformations, in addition to experimentally determined structures, also provides a more accurate estimation for drug efficacy and selectivity. Moreover, obtaining the entire picture of the free energy landscape during ligand binding/unbinding processes is critical in understanding binding mechanisms. Here, we present a Binding Kinetics Toolkit (BKiT) that includes several utilities to analyze trajectories and compute a free energy and kinetics profile. BKiT uses principal component space to generate approximated unbinding or conformational transition coordinates for accurately describing and easily visualizing the molecular motions. We implemented a new partitioning approach to assign indexes along the approximated coordinates that can be used as milestones or microstates. The program can generate input files to run many short classical molecular dynamics simulations and uses milestoning theory to construct the free energy profile and estimate binding residence time. We first validated the method with a host-guest system, aspirin unbinding from β-cyclodextrin, and then applied the protocol to pyrazolourea compounds and cyclin-dependent kinase 8 and cyclin C complexes, a kinase system of pharmacological interest. Overall, our approaches yielded good agreement with published results and suggest ligand design strategies. The computed unbinding free energy landscape also provides a more complete picture of ligand-receptor binding barriers and stable local minima for deepening our understanding of molecular recognition. BKiT is easy to use and has extensible features for future expansion of utilities for postanalysis and calculations. more...

An important part in structure-based drug design is the selection of an appropriate protein structure. It has been revealed that a holo protein structure that contains a well-defined binding site is a much better choice than an apo structure in structure-based drug discovery. Therefore, it is valuable to obtain a holo-like protein conformation from apo structures in the case where no holo structure is available. Meeting the need, we present a robust approach to generate reliable holo-like structures from apo structures by ligand binding site refinement with restraints derived from holo templates with low homology. Our method was tested on a test set of 32 proteins from the DUD-E data set and compared with other approaches. It was shown that our method successfully refined the apo structures toward the corresponding holo conformations for 23 of 32 proteins, reducing the average all-heavy-atom RMSD of binding site residues by 0.48 Å. In addition, when evaluated against all the holo structures in the protein data bank, our method can improve the binding site RMSD for 14 of 19 cases that experience significant conformational changes. Furthermore, our refined structures also demonstrate their advantages over the apo structures in ligand binding mode predictions by both rigid docking and flexible docking and in virtual screening on the database of active and decoy ligands from the DUD-E. These results indicate that our method is effective in recovering holo-like conformations and will be valuable in structure-based drug discovery. more...

The analysis and comparison of protein-binding sites aid various applications in the drug discovery process, e.g., hit finding, drug repurposing, and polypharmacology. Classification of binding sites has been a hot topic for the past 30 years, and many different methods have been published. The rapid development of machine learning computational algorithms, coupled with the large volume of publicly available protein-ligand 3D structures, makes it possible to apply deep learning techniques in binding site comparison. Our method uses a cutting-edge spherical convolutional neural network based on the DeepSphere architecture to learn global representations of protein-binding sites. The model was trained on TOUGH-C1 and TOUGH-M1 data and validated with the ProSPECCTs datasets. Our results show that our model can (1) perform well in protein-binding site similarity and classification tasks and (2) learn and separate the physicochemical properties of binding sites. Lastly, we tested the model on a set of kinases, where the results show that it is able to cluster the different kinase subfamilies effectively. This example demonstrates the method's promise for lead hopping within or outside a protein target, directly based on binding site information. more...

Sequence-specific recognition of transcription factor (TF) binding motifs in the target site of DNA over the vast amount of non-target DNA is of primary importance for the transcriptional regulation of gene expression by the TFs. Binding of TFs to the target site of DNA relies not only on the direct contact formation but also on the structural and conformational features of DNA. Recognition of DNA structural features or shape readout by proteins is an important factor in the context of TF-DNA interaction. Based on the atomistic molecular simulation, here we report the sequence-dependent unique structural features, solvation, and ion-binding properties of biologically relevant AT- and GC-rich human TF binding motifs in DNA. Counterion and water distribution around the motif is found to be sensitive to the motif sequence, which is accompanied with the DNA shape features. The motif sequence affects the electrostatic potential along the grooves, and cytosine methylation alters the DNA shape features. Characteristic solvation properties of TF binding motif DNA fragments infer that an ionic environment and hydration influences are essential to describe TF-DNA interactions. more...

Changing the substrate/cofactor specificity of an enzyme requires multiple mutations at spatially adjacent positions around the substrate pocket. However, this is challenging when solely based on crystal structure information because enzymes undergo dynamic conformational changes during the reaction process. Herein, we proposed a method for estimating the contribution of each amino acid residue to substrate specificity by deploying a phylogenetic analysis with logistic regression. Since this method can estimate the candidate amino acids for mutation by ranking, it is readable and can be used in protein engineering. We demonstrated our concept using redox cofactor conversion of the more...

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli. more...

Insulin is a mainstay of therapy for diabetes mellitus, yet its thermal stability complicates global transportation and storage. Cold-chain transport, coupled with optimized formulation and materials, prevents to some degree nucleation of amyloid and hence inactivation of hormonal activity. These issues hence motivate the design of analogs with increased stability, with a promising approach being single-chain insulins (SCIs), whose C domains (foreshortened relative to proinsulin) resemble those of the single-chain growth factors (IGFs). We have previously demonstrated that optimized SCIs can exhibit native-like hormonal activity with enhanced thermal stability and marked resistance to fibrillation. Here, we describe the crystal structure of an ultrastable SCI (C-domain length 6; sequence EEGPRR) bound to modules of the insulin receptor (IR) ectodomain (N-terminal α-subunit domains L1-CR and C-terminal αCT peptide; "microreceptor" [μIR]). The structure of the SCI-μIR complex, stabilized by an Fv module, was determined using diffraction data to a resolution of 2.6 Å. Remarkably, the αCT peptide (IR-A isoform) "threads" through a gap between the flexible C domain and the insulin core. To explore such threading, we undertook molecular dynamics simulations to 1) compare threaded with unthreaded binding modes and 2) evaluate effects of C-domain length on these alternate modes. The simulations (employing both conventional and enhanced sampling simulations) provide evidence that very short linkers (C-domain length of -1) would limit gap opening in the SCI and so impair threading. We envisage that analogous threading occurs in the intact SCI-IR complex-rationalizing why minimal C-domain lengths block complete activity-and might be exploited to design novel receptor-isoform-specific analogs. more...

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β = 13.5 h) than soluble ACE2 (t½ β = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. Statement of significance: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19. more...

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove more...

Integrin activation is a predominant step for cell-cell and cell-ECM interactions. Talin and Kindlin are mechanosensitive adaptor proteins that bind to the integrin cytoplasmic tail and mediate integrin activation, cytoskeleton rearrangement, and focal adhesion assembly. However, knowledge about how Talin and Kindlin synergistically assist integrin activation remains unclear. Here, we performed so-called "ramp-clamp" SMD simulations, which modeled the mechanosignaling from Kindlin, to investigate the effect of tension on the interaction of the β1 integrin cytoplasmic tail with the Talin-F3 domain. The present results showed that mild but not excessive stretching enhanced the binding of integrin with Talin. This mechanical regulation on integrin affinity to Talin referred to an event cascade, in which under stretching, the integrin cytoplasmic tail adopted allostery in response to the mechanical stimulus, remodeling of integrin in favor of Talin-association ensued, and finally, a stable, close-knit complex was formed. In the cascade, the torsion angle transition of integrin was the cue for the stable interaction of the complex under tensile force. The present work suggested a model for Talin and Kindlin to synergistically activate integrin. It should help understand integrin activation and its mechanochemical regulation mechanism, integrin-related innate cellular immune responses, cell adhesion, cell-cell interaction, and integrin-related drug development. more...

The identification of starting points for compound development is one of the key steps in early-stage drug discovery. Information-rich techniques such as crystallographic fragment screening can potentially increase the efficiency of this step by providing the structural information of the binding mode of the ligands in addition to the mere binding information. Here, we present the crystallographic screening of our 1000-plus-compound F2X-Universal Library against the complex of the yeast spliceosomal Prp8 RNaseH-like domain and the snRNP assembly factor Aar2. The observed 269 hits are distributed over 10 distinct binding sites on the surface of the protein-protein complex. Our work shows that hit clusters from large-scale crystallographic fragment screening campaigns identify known interaction sites with other proteins and suggest putative additional interaction sites. Furthermore, the inherent binding pose validation within the hit clusters may accelerate downstream compound optimization. more...

Peptides are commonly used as therapeutic agents. However, they suffer from easy degradation and instability. Replacing natural by non-natural amino acids can avoid these problems, and potentially improve the affinity towards the target protein. Here, we present a computational pipeline to optimize peptides based on adding non-natural amino acids while improving their binding affinity. The workflow is an iterative computational evolution algorithm, inspired by the PARCE protocol, that performs single-point mutations on the peptide sequence using modules from the Rosetta framework. The modifications can be guided based on the structural properties or previous knowledge of the biological system. At each mutation step, the affinity to the protein is estimated by sampling the complex conformations and applying a consensus metric using various open protein-ligand scoring functions. The mutations are accepted based on the score differences, allowing for an iterative optimization of the initial peptide. The sampling/scoring scheme was benchmarked with a set of protein-peptide complexes where experimental affinity values have been reported. In addition, a basic application using a known protein-peptide complex is also provided. The structure- and dynamic-based approach allows users to optimize bound peptides, with the option to personalize the code for further applications. The protocol, called mPARCE, is available at: https://github.com/rochoa85/mPARCE/. more...

Allosteric regulation of protein activity pervades biology as the "second secret of life." We have been examining the allosteric regulation and mutant reactivation of the tumor suppressor protein p53. We have found that generalizing the definition of allosteric effector to include entire proteins and expanding the meaning of binding site to include the interface of a transcription factor with its DNA to be useful in understanding the modulation of protein activity. Here, we cast the variable regions of p53 isoforms as allosteric regulators of p53 interactions with its consensus DNA. We implemented molecular dynamics simulations and our lab's new techniques of molecular dynamics (MD) sectors and MD-Markov state models to investigate the effects of nine naturally occurring splice variant isoforms of p53. We find that all of the isoforms differ from wild type in their dynamic properties and how they interact with the DNA. We consider the implications of these findings on allostery and cancer treatment. more...

Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs more...

The COVID-19 pandemic has caused significant social and economic disruption across the globe. Cellular entry of SARS-CoV-2 into the human body is mediated via binding of the Receptor Binding Domain (RBD) on the viral Spike protein (SARS-CoV-2 RBD) to Angiotensin-Converting Enzyme 2 (ACE2) expressed on host cells. Molecules that can disrupt ACE2:RBD interactions are attractive therapeutic candidates to prevent virus entry into human cells. A computational strategy that combines our Peptide Binding Design (PepBD) algorithm with atomistic molecular dynamics simulations was used to design new inhibitory peptide candidates via sequence iteration starting with a 23-mer peptide, referred to as SBP1. SBP1 is derived from a region of the ACE2 Peptidase Domain α1 helix that binds to the SARS-CoV-2 RBD of the initial Wuhan-Hu-1 strain. Three peptides demonstrated a solution-phase RBD-binding dissociation constant in the micromolar range during tryptophan fluorescence quenching experiments, one peptide did not bind, and one was insoluble at micromolar concentrations. However, in competitive ELISA assays, none of these peptides could outcompete ACE2 binding to SARS-CoV-2-RBD up to concentrations of 50 μM, similar to the parent SBP1 peptide which also failed to outcompete ACE2:RBD binding. Molecular dynamics simulations suggest that P4 would have a good binding affinity for the RBD domain of Beta-B.1.351, Gamma-P.1, Kappa-B.1.617.1, Delta-B.1.617.2, and Omicron-B.1.1.529 variants, but not the Alpha variant. Consistent with this, P4 bound Kappa-B.1.617.1 and Delta-B.1.617.2 RBD with micromolar affinity in tryptophan fluorescence quenching experiments. Collectively, these data show that while relatively short unstructured peptides can bind to SARS-CoV-2 RBD with moderate affinity, they are incapable of outcompeting the strong interactions between RBD and ACE2. more...

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications. more...

Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein-protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow addressing two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands' gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity. more...

Knowledge of the transcription factor binding landscape (TFBL) is necessary to analyze gene regulatory networks for important agronomic traits. However, a low-cost and high-throughput in vivo chromatin profiling method is still lacking in plants. Here, we developed a transient and simplified cleavage under targets and tagmentation (tsCUTTag) that combines transient expression of transcription factor proteins in protoplasts with a simplified CUTTag without nucleus extraction. Our tsCUTTag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP-seq. Furthermore, we developed a strategy combining tsCUTTag with machine learning, which has great potential for profiling the TFBL across plant development. more...

Understanding the protein-RNA interaction mechanism can help us to further explore various biological processes. The experimental techniques still have some limitations, such as the high cost of economy and time. Predicting protein-RNA-binding sites by using computational methods is an excellent research tool. Here, we developed a universal method for predicting protein-specific RNA-binding sites, so one general model for a given protein was constructed on a fixed dataset by fusing the data of different experimental techniques. At the same time, information theory was employed to characterize the sequence conservation of RNA-binding segments. Conversation difference profiles between binding and nonbinding segments were constructed by information entropy (IE), which indicates a significant difference. Finally, the 19 proteins-specific models based on random forest (RF) were built based on IE encoding. The performance on the independent datasets demonstrates that our method can obtain competitive results when compared with the current best prediction model. more...

Background:Cardiovascular disease (CVD) is a leading cause of death worldwide. It is predicted that approximately 23.6 million people will die from CVDs annually by 2030. Therefore, there is a great need for an effective therapeutic approach to combat this disease. The European Cardiovascular Target Discovery (CarTarDis) consortium identified Oncostatin M (OSM) as a po-tential therapeutic target for atherosclerosis. The benefits of modulating OSM - an interleukin (IL)-6 family cytokine - have since been studied for multiple indications. However, as decades of high at-trition rates have stressed, the success of a drug target is determined by the fine balance between benefits and the risk of adverse events. Safety issues should therefore not be overlooked.Objective:In this review, a risk/benefit analysis is performed on OSM inhibition in the context of atherosclerosis treatment. First, OSM signaling characteristics and its role in atherosclerosis are de-scribed. Next, an overview of in vitro, in vivo, and clinical findings relating to both the benefits and risks of modulating OSM in major organ systems is provided. Based on OSM’s biological function and expression profile as well as drug intervention studies, safety concerns of inhibiting this target have been identified, assessed, and ranked for the target population.Conclusion:While OSM may be of therapeutic value in atherosclerosis, drug development should also focus on de-risking the herein identified major safety concerns: tissue remodeling, angiogene-sis, bleeding, anemia, and NMDA- and glutamate-induced neurotoxicity. Close monitoring and/or exclusion of patients with various comorbidities may be required for optimal therapeutic benefit. more...

The adaptability of proteins to their work environments is fundamental for cellular life. Here, we describe how the hemopexin-like domain of the multifunctional blood glycoprotein vitronectin binds Ca more...

The human VPS10 domain-containing receptor SorCS3 belongs to the Vps10p-domain receptor family and is an important receptor for regulating normal cellular functions via protein sorting. Here, we determined the cryo-EM structure of the full-length SorCS3 receptor and further found that there were at least three distinct conformations (monomer, M-shaped dimer and N-shaped dimer) of SorCS3 in the apo state. The differences between the two dimer conformations were caused by PKD1-2 assembly. In contrast to its homologous proteins, the conserved residues GLN198, ARG678, TYR430, GLU1020 and ASP1024 may be key points for its dimerization and for protein/polypeptide binding. These results showed the structural details of apo-SorCS3, which provides a foundation for elucidating the mechanism of protein sorting. more...

Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1.To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction.A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico.Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII.FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1. more...

Inhibition of the bromodomain of the CREB (cyclic-AMP response element-binding protein) binding protein (CBP) is a particularly promising new therapeutic approach for cancer. Benzimidazole derivatives CCS1477 and its analogues (8 and 9) are highly potent and selective CBP bromodomain inhibitors, with K more...

Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses. more...

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code. more...

A growing number of G-protein-coupled receptor (GPCR) structures reveal novel transmembrane lipid-exposed allosteric sites. Ligands must first partition into the surrounding membrane and take lipid paths to these sites. Remarkably, a significant part of the bound ligands appears exposed to the membrane lipids. The experimental structures do not usually account for the surrounding lipids, and their apparent contribution to ligand access and binding is often overlooked and poorly understood. Using classical and enhanced molecular dynamics simulations, we show that membrane lipids are critical in the access and binding of ORG27569 and its analogs at the transmembrane site of cannabinoid CB1 receptor. The observed differences in the binding affinity and cooperativity arise from the functional groups that interact primarily with lipids. Our results demonstrate the significance of incorporating membrane lipids as an integral component of transmembrane sites for accurate characterization, binding-affinity calculations, and lead optimization in drug discovery. more...

Influenza polymerase (FluPol) plays a key role in the viral infection cycle by transcribing and replicating the viral genome. FluPol is a multifunctional, heterotrimeric enzyme with cap-binding, endonuclease, RNA-dependent RNA polymerase and polyadenylation activities. It performs its functions in the context of the viral ribonucleoprotein particle (RNP), wherein the template viral RNA is coated by multiple copies of viral nucleoprotein. Moreover, it interacts with a number of host proteins that are essential cofactors and, consequently, adaptive mutations in the polymerase are required for crossing the avian-human species barrier. In this review, we show how mechanistic understanding of how FluPol performs its multiple functions has greatly advanced over the last decade through determination of high-resolution structures by X-ray crystallography and cryo-electron microscopy. These have revealed not only the detailed architecture of FluPol but highlighted the remarkably conformational flexibility that is inherent to its functioning as a dynamic RNA synthesis machine. Structural studies are also underpinning current attempts to develop next-generation anti-influenza drugs that directly target FluPol. more...

The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin. more...

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to β-arrestins (βarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor. more...

Antibodies are key proteins produced by the immune system to target pathogen proteins termed antigens via specific binding to surface regions called epitopes. Given an antigen and the sequence of an antibody the knowledge of the epitope is critical for the discovery and development of antibody based therapeutics. In this work, we present a computational protocol that uses template-based modeling and docking to predict epitope residues. This protocol is implemented in three major steps. First, a template-based modeling approach is used to build the antibody structures. We tested several options, including generation of models using AlphaFold2. Second, each antibody model is docked to the antigen using the fast Fourier transform (FFT) based docking program PIPER. Attention is given to optimally selecting the docking energy parameters depending on the input data. In particular, the van der Waals energy terms are reduced for modeled antibodies relative to x-ray structures. Finally, ranking of antigen surface residues is produced. The ranking relies on the docking results, that is, how often the residue appears in the docking poses' interface, and also on the energy favorability of the docking pose in question. The method, called PIPER-Map, has been tested on a widely used antibody-antigen docking benchmark. The results show that PIPER-Map improves upon the existing epitope prediction methods. An interesting observation is that epitope prediction accuracy starting from antibody sequence alone does not significantly differ from that of starting from unbound (i.e., separately crystallized) antibody structure. more...

The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots. more...

Systematic and quantitative analysis of the reliability of formally exact methods that

Hypersensitive regulation of cellular hypoxic response relies on cooperative displacement of one disordered protein (HIF-1α) by another disordered protein (CITED2) from the target in a negative feedback loop. Considering the weak intramolecule coupling in disordered proteins, the molecular mechanism of high cooperativity in the molecular displacement event remains elusive. Herein, we show that disordered proteins utilize a "self-effected allostery" mechanism to achieve high binding cooperativity. Different from the conventional allostery mechanisms shown by many structured or disordered proteins, this mechanism utilizes one part of the disordered protein as the effector to trigger the allosteric coupling and enhance the binding of the remaining part of the same disordered protein, contributing to high cooperativity of the displacement event. The conserved charge motif of CITED2 is the key determinant of the molecular displacement event by serving as the effector of allosteric coupling. Such self-effected allostery provides an efficient strategy to achieve high cooperativity in the molecular events involving disordered proteins. more...

SH2 domains have been recognized as promising targets for various human diseases. However, targeting SH2 domains with phosphopeptides or small-molecule inhibitors derived from bioisosteres of the phosphate group is still challenging. Identifying novel bioisosteres of the phosphate group to achieve favorable more...

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning. more...