How fingers affect folding of a thumb: Inter-subdomain cooperation in the folding of SARS-CoV-2 RdRp protein.

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.

Shifting paradigms: The promise of allosteric inhibitors against dengue virus protease.

Dengue, a mosquito-borne viral infection caused by the dengue virus (DENV), is a global health challenge. Annually, approximately 400 million cases are reported worldwide, signaling a persistent upward trend from previous years and projected a manifold increase in the future. There is a growing need for innovative and integrated approaches aimed at effective disease management. In this regard, scientific efforts are underway to find a new antiviral inhibitor that is desperately needed due to the growing prevalence of dengue, along with inadequate vector control and few vaccinations. The NS2B-NS3 protease complex within the DENV genome holds significant importance, making it an attractive target for potential interventions. Many competitive inhibitors are not clinically relevant even after extensive study, and these early hits are often not followed up to viable leads. The current focus is on exploring alternative target sites for developing effective anti-dengue compounds, resulting in the identification of various allosteric sites in recent years. While previous reviews have extensively covered active site inhibitors, this is to the best of our knowledge the first comprehensive review discussing the allosteric sites and allosteric inhibitors in greater detail. The present survey may assist researchers in understanding the key aspects and identifying new antagonists targeting the allosteric site of DENV protease.

Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET.

Cyclic nucleotide-binding domain (CNBD) ion channels play crucial roles in cellular-signaling and excitability and are regulated by the direct binding of cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP). However, the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood. The prokaryotic CNBD channel SthK offers a valuable model for investigating this allosteric mechanism. In this study, we investigated the conformational dynamics and energetics of the SthK C-terminal region using a combination of steady-state and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) experiments. We engineered donor-acceptor pairs at specific sites within a SthK C-terminal fragment by incorporating a fluorescent noncanonical amino acid donor and metal ion acceptors. Measuring tmFRET with fluorescence lifetimes, we determined intramolecular distance distributions in the absence and presence of cAMP or cGMP. The probability distributions between conformational states without and with ligand were used to calculate the changes in free energy ( ΔG ) and differences in free energy change ( ΔΔG ) in the context of a simple four-state model. Our findings reveal that cAMP binding produces large structural changes, with a very favorable ΔΔG . In contrast to cAMP, cGMP behaved as a partial agonist and only weakly promoted the active state. Furthermore, we assessed the impact of protein oligomerization and ionic strength on the structure and energetics of the conformational states. This study demonstrates the effectiveness of time-resolved tmFRET in determining the conformational states and the ligand-dependent energetics of the SthK C-terminal region.

Mechanistic basis of activation and inhibition of Protein Disulfide Isomerase by allosteric antithrombotic compounds.

BACKGROUND: Protein Disulfide Isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development. OBJECTIVES: To define the mechanism of action of two allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a. METHODS: A multi-pronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single turnover kinetics, and site-specific mutagenesis. RESULTS: PDI is a thiol-isomerase consisting of two catalytic a-domains and two inactive b-domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of nine FRET sensors, we show that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, FRET data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme-substrate complex. CONCLUSIONS: This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a, and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.

Drugs Targeting Sirtuin 2 Exhibit Broad-Spectrum Anti-Infective Activity.

Direct-acting anti-infective drugs target pathogen-coded gene products and are a highly successful therapeutic paradigm. However, they generally target a single pathogen or family of pathogens, and the targeted organisms can readily evolve resistance. Host-targeted agents can overcome these limitations. One family of host-targeted, anti-infective agents modulate human sirtuin 2 (SIRT2) enzyme activity. SIRT2 is one of seven human sirtuins, a family of NAD+-dependent protein deacylases. It is the only sirtuin that is found predominantly in the cytoplasm. Multiple, structurally distinct SIRT2-targeted, small molecules have been shown to inhibit the replication of both RNA and DNA viruses, as well as intracellular bacterial pathogens, in cell culture and in animal models of disease. Biochemical and X-ray structural studies indicate that most, and probably all, of these compounds act as allosteric modulators. These compounds appear to impact the replication cycles of intracellular pathogens at multiple levels to antagonize their replication and spread. Here, we review SIRT2 modulators reported to exhibit anti-infective activity, exploring their pharmacological action as anti-infectives and identifying questions in need of additional study as this family of anti-infective agents advances to the clinic.

Cooperative Substructure and Energetics of Allosteric Regulation of the Catalytic Core of the E3 Ubiquitin Ligase Parkin by Phosphorylated Ubiquitin.

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise control of its catalytic activity. Though its allosterically controlled structural reorganization has been extensively characterized by crystallography, the energetics and mechanisms of allosteric regulation of Parkin are much less well understood. Allostery is fundamentally linked to the energetics of the cooperative (sub)structure of the protein. Herein, we examine the mechanism of allosteric activation by phosphorylated ubiquitin binding to the enzymatic core of Parkin, which lacks the antagonistic Ubl domain. In this way, the allosteric effects of the agonist phosphorylated ubiquitin can be isolated. Using native-state hydrogen exchange monitored by mass spectrometry, we find that the five structural domains of the core of Parkin are energetically distinct. Nevertheless, association of phosphorylated ubiquitin destabilizes structural elements that bind the ubiquitin-like domain antagonist while promoting the dissociation of the catalytic domain and energetically poises the protein for transition to the fully activated structure.

MEF-AlloSite: an accurate and robust Multimodel Ensemble Feature selection for the Allosteric Site identification model.

A crucial mechanism for controlling the actions of proteins is allostery. Allosteric modulators have the potential to provide many benefits compared to orthosteric ligands, such as increased selectivity and saturability of their effect. The identification of new allosteric sites presents prospects for the creation of innovative medications and enhances our comprehension of fundamental biological mechanisms. Allosteric sites are increasingly found in different protein families through various techniques, such as machine learning applications, which opens up possibilities for creating completely novel medications with a diverse variety of chemical structures. Machine learning methods, such as PASSer, exhibit limited efficacy in accurately finding allosteric binding sites when relying solely on 3D structural information.Scientific ContributionPrior to conducting feature selection for allosteric binding site identification, integration of supporting amino-acid-based information to 3D structural knowledge is advantageous. This approach can enhance performance by ensuring accuracy and robustness. Therefore, we have developed an accurate and robust model called Multimodel Ensemble Feature Selection for Allosteric Site Identification (MEF-AlloSite) after collecting 9460 relevant and diverse features from the literature to characterise pockets. The model employs an accurate and robust multimodal feature selection technique for the small training set size of only 90 proteins to improve predictive performance. This state-of-the-art technique increased the performance in allosteric binding site identification by selecting promising features from 9460 features. Also, the relationship between selected features and allosteric binding sites enlightened the understanding of complex allostery for proteins by analysing selected features. MEF-AlloSite and state-of-the-art allosteric site identification methods such as PASSer2.0 and PASSerRank have been tested on three test cases 51 times with a different split of the training set. The Student's t test and Cohen's D value have been used to evaluate the average precision and ROC AUC score distribution. On three test cases, most of the p-values ( < 0.05 ) and the majority of Cohen's D values ( > 0.5 ) showed that MEF-AlloSite's 1-6% higher mean of average precision and ROC AUC than state-of-the-art allosteric site identification methods are statistically significant.

Dynamic sampling of a surveillance state enables DNA proofreading by Cas9.

CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled in vitro, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this "surveillance state" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.

Impact of replicas and simulation length on in silico behaviors of a protein domain.

Molecular dynamics (MD) simulations are a powerful tool for life sciences, valuable for their ability to capture atomic-level behavior of molecules over time. To advance knowledge on reasonable timescales, researchers must optimize the amount of useful information extracted from simulation data while frugally managing computational resources. They must balance trajectory lengths and number of replicas, with the aim of maximizing conformational sampling. Identifying this balance is not always intuitive, and lack of standardization among researchers produces variability in results from MD measurements. We investigate how changes in simulation length and replica numbers impact this variability. Using a 231-residue domain, we compare measurements from independent trajectories to a benchmark trajectory of 3x1000-ns replicates. We simulate 27 protein-ligand complexes, allowing us to compare ligand-specific rankings of complexes across replicas. We reveal that some MD measurements are reliably ranked by single trajectories, while others are not. We uncover similar variability in the effects of trajectory lengths on measurements. Our findings suggest that a one-size-fits-all approach to MD simulations is not ideal, and depending on the intended measurements and research question, it is sometimes advantageous to prioritize longer trajectories over multiple replicas. This work provides important considerations for researchers while designing simulation studies.

SecM leader peptide as an allosteric translation inhibitor: a molecular dynamics study.

The SecM leader peptide regulates translation of the SecA protein, being a part of the Sec translocase, that reversibly arrests the ribosome. In the present study the structure of the SecM complex with the E. coli A/A,P/P-ribosome was obtained by means of docking and molecular dynamics simulation methods. It has been established that binding of the SecM leader peptide in the nascent peptide exit tunnel leads to a turn of the aminoacylating proline residue away from the C-terminal SecM glycine residue, which is adverse to the peptidyltransferase reaction. Besides, the SecM binding leads to a disturbance of the A-tRNA contacts with the tip of the H38 helix of the 23S rRNA (the A-site finger, ASF) and ribosomal protein uL16. Allosteric interrelation between these events has been proved by a construction of networks of concerted changes in non-covalent interactions throughout the whole ribosome, whereupon the A1614 and A751 residues of the 23S rRNA in the exit tunnel that formed stacking interactions with the SecM residues during the MD simulations, were found to be the principal triggers, inducing crucial alterations in the A-tRNA binding. The allosteric signal from the SecM peptide to the ASF, according to our model, is transmitted through ribosomal protein uL22, and there is reason to believe that this sensor is used not only by the SecM leader peptide, but also by other peptides that cause translation arrest.

Amyloid-Driven Allostery.

The fields of allostery and amyloid-related pathologies, such as Parkinson's disease (PD), have been extensively explored individually, but less is known about how amyloids control allostery. Recent advancements have revealed that amyloids can drive allosteric effects in both intrinsically disordered proteins, such as alpha-synuclein (αS), and multi-domain signaling proteins, such as protein kinase A (PKA). Amyloid-driven allostery plays a central role in explaining the mechanisms of gain-of-pathological-function mutations in αS (e.g. E46K, which causes early PD onset) and loss-of-physiological-function mutations in PKA (e.g. A211D, which predisposes to tumors). This review highlights allosteric effects of disease-related mutations and how they can cause exposure of amyloidogenic regions, leading to amyloids that are either toxic or cause aberrant signaling. We also discuss multiple potential modulators of these allosteric effects, such as MgATP and kinase substrates, opening future opportunities to improve current pharmacological interventions against αS and PKA-related pathologies. Overall, we show that amyloid-driven allosteric models are useful to explain the mechanisms underlying disease-related mutations.

Cooperative folding as a molecular switch in an evolved antibody binder.

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody binding. Hence, these variants contains molecular "on/off" switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase.

Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.

Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR.

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C-13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

Dynamic allostery drives autocrine and paracrine TGF-ß signaling.

TGF-ß, essential for development and immunity, is expressed as a latent complex (L-TGF-ß) non-covalently associated with its prodomain and presented on immune cell surfaces by covalent association with GARP. Binding to integrin αvß8 activates L-TGF-ß1/GARP. The dogma is that mature TGF-ß must physically dissociate from L-TGF-ß1 for signaling to occur. Our previous studies discovered that αvß8-mediated TGF-ß autocrine signaling can occur without TGF-ß1 release from its latent form. Here, we show that mice engineered to express TGF-ß1 that cannot release from L-TGF-ß1 survive without early lethal tissue inflammation, unlike those with TGF-ß1 deficiency. Combining cryogenic electron microscopy with cell-based assays, we reveal a dynamic allosteric mechanism of autocrine TGF-ß1 signaling without release where αvß8 binding redistributes the intrinsic flexibility of L-TGF-ß1 to expose TGF-ß1 to its receptors. Dynamic allostery explains the TGF-ß3 latency/activation mechanism and why TGF-ß3 functions distinctly from TGF-ß1, suggesting that it broadly applies to other flexible cell surface receptor/ligand systems.

Navigating the complexity of p53-DNA binding: implications for cancer therapy.

Abstract: The tumor suppressor protein p53, a transcription factor playing a key role in cancer prevention, interacts with DNA as its primary means of determining cell fate in the event of DNA damage. When it becomes mutated, it opens damaged cells to the possibility of reproducing unchecked, which can lead to formation of cancerous tumors. Despite its critical role, therapies at the molecular level to restore p53 native function remain elusive, due to its complex nature. Nevertheless, considerable information has been amassed, and new means of investigating the problem have become available. Objectives: We consider structural, biophysical, and bioinformatic insights and their implications for the role of direct and indirect readout and how they contribute to binding site recognition, particularly those of low consensus. We then pivot to consider advances in computational approaches to drug discovery. Materials and methods: We have conducted a review of recent literature pertinent to the p53 protein. Results: Considerable literature corroborates the idea that p53 is a complex allosteric protein that discriminates its binding sites not only via consensus sequence through direct H-bond contacts, but also a complex combination of factors involving the flexibility of the binding site. New computational methods have emerged capable of capturing such information, which can then be utilized as input to machine learning algorithms towards the goal of more intelligent and efficient de novo allosteric drug design. Conclusions: Recent improvements in machine learning coupled with graph theory and sector analysis hold promise for advances to more intelligently design allosteric effectors that may be able to restore native p53-DNA binding activity to mutant proteins. Clinical relevance: The ideas brought to light by this review constitute a significant advance that can be applied to ongoing biophysical studies of drugs for p53, paving the way for the continued development of new methodologies for allosteric drugs. Our discoveries hold promise to provide molecular therapeutics which restore p53 native activity, thereby offering new insights for cancer therapies. Graphical Abstract: Structural representation of the p53 DBD (PDBID 1TUP). DNA consensus sequence is shown in gray, and the protein is shown in blue. Red beads indicate hotspot residue mutations, green beads represent DNA interacting residues, and yellow beads represent both.

Discovery of potent allosteric antibodies inhibiting EGFR.

In this work, we report the discovery of potent anti-epidermal growth factor receptor (EGFR) allosteric heavy-chain antibodies by combining camelid immunization and fluorescence-activated cell sorting (FACS). After immunization and yeast surface display library construction, allosteric clones were obtained by introducing the labeled EGF Fc fusion protein as an additional criterion for FACS. This sorting method enabled the identification of 11 heavy-chain antibodies that did not compete with the orthosteric ligand EGF for the binding to EGFR. These antibodies bind to a triple-negative breast cancer cell line expressing EGFR with affinities in the picomolar to nanomolar range. Those camelid-derived antibodies also exhibit interesting properties by modulating EGFR affinity for EGF. Moreover, they are also able to inhibit EGF-induced downstream signaling pathways. In particular, we identified one clone that is more potent than the approved blocking antibody cetuximab in inhibiting both PI3K/AKT and MAPK/ERK pathways. Our results suggest that allosteric antibodies may be potential new modalities for therapeutics.

Free energy landscape of the PI3Kα C-terminal activation.

The gene PIK3CA, encoding the catalytic subunit p110α of PI3Kα, is the second most frequently mutated gene in cancer, with the highest frequency oncogenic mutants occurring in the C-terminus of the kinase domain. The C-terminus has a dual function in regulating the kinase, playing a putative auto-inhibitory role for kinase activity and being absolutely essential for binding to the cell membrane. However, the molecular mechanisms by which these C-terminal oncogenic mutations cause PI3Kα overactivation remain unclear. To understand how a spectrum of C-terminal mutations of PI3Kα alter kinase activity compared to the WT, we perform unbiased and biased Molecular Dynamics simulations of several C-terminal mutants and report the free energy landscapes for the C-terminal "closed-to-open" transition in the WT, H1047R, G1049R, M1043L and N1068KLKR mutants. Results are consistent with HDX-MS experimental data and provide a molecular explanation why H1047R and G1049R reorient the C-terminus with a different mechanism compared to the WT and M1043L and N1068KLKR mutants. Moreover, we show that in the H1047R mutant, the cavity, where the allosteric ligands STX-478 and RLY-2608 bind, is more accessible contrary to the WT. This study provides insights into the molecular mechanisms underlying activation of oncogenic PI3Kα by C-terminal mutations and represents a valuable resource for continued efforts in the development of mutant selective inhibitors as therapeutics.

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

A platform for the early selection of non-competitive antibody-fragments from yeast surface display libraries.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.