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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.

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NMDA receptors (NMDARs) are glutamate-gated ion channels playing a central role in synaptic transmission and plasticity. NMDAR dysregulation is linked to various neuropsychiatric disorders. This is particularly true for GluN2B-containing NMDARs (GluN2B-NMDARs), which have major pro-cognitive, but also pro-excitotoxic roles, although their exact involvement in these processes remains debated. Traditional GluN2B-selective antagonists suffer from slow and irreversible effects, limiting their use in native tissues. We therefore developed OptoNAM-3, a photoswitchable negative allosteric modulator selective for GluN2B-NMDARs. OptoNAM-3 provided light-induced reversible inhibition of GluN2B-NMDAR activity with precise temporal control both in vitro and in vivo on the behavior of freely moving Xenopus tadpoles. When bound to GluN2B-NMDARs, OptoNAM-3 displayed remarkable red-shifting of its photoswitching properties allowing the use of blue light instead of UV light to turn-off its activity, which we attributed to geometric constraints imposed by the binding site onto the azobenzene moiety of the ligand. This study therefore highlights the importance of the binding site in shaping the photochemical properties of azobenzene-based photoswitches. In addition, by enabling selective, fast, and reversible photocontrol of native GluN2B-NMDARs with in vivo compatible photochemical properties (visible light), OptoNAM-3 should be a useful tool for the investigation of the GluN2B-NMDAR physiology in native tissues.

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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.

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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.

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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.

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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.

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Multi-domain enzymes can be regulated by both inter-domain interactions and structural features intrinsic to the catalytic domain. The tyrosine phosphatase SHP2 is a quintessential example of a multi-domain protein that is regulated by inter-domain interactions. This enzyme has a protein tyrosine phosphatase (PTP) domain and two phosphotyrosine-recognition domains (N-SH2 and C-SH2) that regulate phosphatase activity through autoinhibitory interactions. SHP2 is canonically activated by phosphoprotein binding to the SH2 domains, which causes large inter-domain rearrangements, but autoinhibition can also be disrupted by disease-associated mutations. Many details of the SHP2 activation mechanism are still unclear, the physiologically-relevant active conformations remain elusive, and hundreds of human variants of SHP2 have not been functionally characterized. Here, we perform deep mutational scanning on both full-length SHP2 and its isolated PTP domain to examine mutational effects on inter-domain regulation and catalytic activity. Our experiments provide a comprehensive map of SHP2 mutational sensitivity, both in the presence and absence of inter-domain regulation. Coupled with molecular dynamics simulations, our investigation reveals novel structural features that govern the stability of the autoinhibited and active states of SHP2. Our analysis also identifies key residues beyond the SHP2 active site that control PTP domain dynamics and intrinsic catalytic activity. This work expands our understanding of SHP2 regulation and provides new insights into SHP2 pathogenicity.

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G protein-coupled receptors (GPCRs) exemplify sophisticated allosteric communication, transducing extracellular signals through ligand-induced structural rearrangements that resonate through the molecular scaffold. Despite extensive study, the biophysical underpinnings of how conformational changes spread remain unclear. This work employs a novel physics-based framework to characterize the role of energy dissipation in directing intramolecular signaling pathways. By modeling each residue as a network of coupled oscillators, we generate a localization landscape depicting the vibrational energy distribution throughout the protein scaffold. Quantifying directional energy flux between residues reveals distinct pathways for energy and information transfer, illuminating sequences of allosteric communication. Our analysis of CB1 and CCR5 crystal structures unveils an anisotropic pattern of energy dissipation aligning with key functional dynamics, such as activation-related conformational changes. These anisotropic patterns of vibrational energy flow constitute pre-configured channels for allosteric signaling. Elucidating the relationship between structural topology and energy dissipation patterns provides key insights into the thermodynamic drivers of conformational signaling. This methodology significantly advances our mechanistic understanding of allostery in GPCRs and presents a broadly applicable approach for rationally dissecting allosteric communication pathways, with potential implications for structure-based drug design targeting these critical receptors.

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Motional properties of proteins govern recognition, catalysis, and regulation. The dynamics of tightly interacting residues can form intramolecular dynamic networks, dependencies fine-tuned by evolution to optimize a plethora of functional aspects. The constructive interaction of residues from different proteins to assemble intermolecular dynamic networks is a similarly likely case but has escaped thorough experimental assessment due to interfering association/dissociation dynamics. Here, we use fast-MAS solid-state 15N R1ρ NMR relaxation dispersion aided by molecular-dynamics simulations to mechanistically assess the hierarchy of individual µs timescale motions arising from a crystal-crystal contact, in the absence of translational motion. In contrast to the monomer, where particular mutations entail isolated perturbations, specific intermolecular interactions couple the motional properties between distant residues in the same protein. The mechanistic insights obtained from this conceptual work may improve our understanding on how intramolecular allostery can be tuned by intermolecular interactions via assembly of dynamic networks from previously isolated elements.

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The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.

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BACKGROUND: Testosterone, estradiol, and dihydrotestosterone share common ligand binding sites on sex hormone binding globulin and albumin. It is unknown whether and how changes in testosterone, dihydrotestosterone, and estradiol concentrations during testosterone replacement therapy affect free testosterone fraction. OBJECTIVE: To determine the effect of changes in testosterone, dihydrotestosterone, and estradiol concentrations on free testosterone fraction during testosterone replacement therapy of men with hypogonadism. METHODS: Using data from the Testosterone Trials, we assessed the association of changes in total testosterone, estradiol, and dihydrotestosterone concentrations over 12 months of testosterone replacement therapy with changes in free testosterone fraction, measured using equilibrium dialysis. We used random forests to evaluate the associations of predicted mean changes in free testosterone fraction with changes in circulating concentrations of each hormone at low, mean, or high change in the other two hormones. RESULTS: Testosterone replacement therapy not only increased total testosterone, dihydrotestosterone, estradiol, and free testosterone concentrations, but also the percent free testosterone, even though sex hormone binding globulin levels did not change. The predicted changes in free testosterone fraction during testosterone replacement therapy exhibited a non-linear relationship with changes in each of total testosterone, dihydrotestosterone, and estradiol concentrations. Greater increases in testosterone, dihydrotestosterone, and estradiol levels during testosterone replacement therapy were each associated with higher model-predicted percent free testosterone. Substantially smaller changes in molar concentrations of estradiol and dihydrotestosterone had a greater effect on percent free testosterone than those in testosterone. CONCLUSION: During testosterone replacement therapy of men with hypogonadism, changes in testosterone, dihydrotestosterone, and estradiol concentrations each altered percent free testosterone non-linearly. Small changes in estradiol concentrations exerted much larger effect on the free testosterone fraction than testosterone and dihydrotestosterone, suggesting complex interactions of the three hormones with the binding proteins. Assessment of changes in free testosterone during testosterone replacement therapy should include consideration of changes in all three hormones.

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Most cancer cells exhibit high glycolysis rates under conditions of abundant oxygen. Maintaining a stable glycolytic rate is critical for cancer cell growth as it ensures sufficient conversion of glucose carbons to energy, biosynthesis, and redox balance. Here we deciphered the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway. Knocking down or knocking out PKM2 induced a thermodynamic equilibration in the glycolytic pathway, characterized by the reciprocal changes of the Gibbs free energy (ΔG) of the reactions catalyzed by PFK1 and PK, leading to a less exergonic PFK1-catalyzed reaction and a more exergonic PK-catalyzed reaction. The changes in the ΔGs of the two reactions cause the accumulation of intermediates, including the substrate PEP (the substrate of PK), in the segment between PFK1 and PK. The increased concentration of PEP in turn increased PK activity in the glycolytic pathway. Thus, the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway maintains the reciprocal relationship between PK concentration and its substrate PEP concentration, by which, PK activity in the glycolytic pathway can be stabilized and effectively counteracts the effect of PKM2 KD or KO on glycolytic rate. In line with our previous reports, this study further validates the roles of the thermodynamics of the glycolytic pathway in stabilizing glycolysis in cancer cells. Deciphering the interaction between glycolytic enzymes and the thermodynamics of the glycolytic pathway will promote a better understanding of the flux control of glycolysis in cancer cells.

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In recent decades, taste sensors have been increasingly utilized to assess the taste of oral medicines, particularly focusing on bitterness, a major obstacle to patient acceptance and adherence. This objective and safe method holds promise for enhancing the development of patient-friendly medicines in pharmaceutical companies. This review article introduces its application in measuring the intensity of bitterness in medicine, confirming the achievement of taste masking, distinguishing taste differences between branded and generic medicines, and identifying substances to suppress bitterness in target medicines. Another application of the sensor is to predict a significant increase in bitterness when medicine is taken with certain foods/beverages or concomitant medication. Additionally, to verify the sensor's predictability, a significant correlation has been demonstrated between the output of a bitter-sensitive sensor designed for drug bitterness (BT0) and the bitterness responses of the human taste receptor hT2R14 from BitterDB (huji.ac.il). As a recent advancement, a novel taste sensor equipped with lipid/polymer membranes modified by 3-Br-2,6-dihydroxybenzoic acid (2,6-DHBA), based on the concept of allostery, is introduced. This sensor successfully predicts the bitterness of non-charged pharmaceuticals with xanthine skeletons, such as caffeine or related compounds. Finally, the future prospects of taste sensors are discussed.

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N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.

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In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

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The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.

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Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes.

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Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

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The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter cell signaling or protein function. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling.

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Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.

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