Low-affinity Ligands Research Articles

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells.

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo , we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbβ3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Pharmacological inhibition of HPK1 synergizes with PD-L1 blockade to provoke antitumor immunity against tumors with low antigenicity

Immune checkpoint inhibitors have significantly transformed the landscape of cancer therapy. Nevertheless, while these inhibitors are highly effective for certain patient groups, many do not benefit due to primary or acquired resistance. Specifically, these treatments often lack sufficient therapeutic efficacy against cancers with low antigenicity. Thus, the development of an effective strategy to overcome cancers with low antigenicity is imperative for advancing next-generation cancer immunotherapy. Here, we show that small molecule inhibitor of hematopoietic progenitor kinase 1 (HPK1) combined with programmed cell death ligand 1 (PD-L1) blockade can enhance T-cell response to tumor with low antigenicity. We found that treatment of OT-1 splenocytes with HPK1 inhibitor enhanced the activation of signaling molecules downstream of T-cell receptor provoked by low-affinity-antigen stimulation. Using an in vivo OT-1 T-cell transfer model, we demonstrated that combining the HPK1 inhibitor with the anti-PD-L1 antibody significantly suppressed the growth of tumors expressing low-affinity altered peptide ligand of chicken ovalbumin, while anti-PD-L1 antibody monotherapy was ineffective. Our findings offer crucial insights into the potential for overcoming tumors with low antigenicity by combining conventional immune checkpoint inhibitors with HPK1 inhibitor.

Abstract 4365: Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein

Abstract Glycosylation is a tightly regulated post-translational modification where structured sugar groups are added to protein amino acid backbones, most commonly on asparagine for N-glycosylation. The glycosylation-regulating metabolic enzyme uridine diphosphate-glucose pyrophosphorylase 2 (UGP2) is required in oncogene-transformed cancer cells, but not in normal cells. UGP2 heavily regulates glycosylation of epidermal growth factor receptor (EGFR) at asparagine 361 (N361), a key site near the ligand binding domain. EGFR is a transmembrane tyrosine kinase receptor that promotes cell proliferation via growth cascades. Mutations and amplifications in EGFR account for a significant fraction of non-small cell lung carcinomas and breast adenocarcinoma, with L858R being the most common oncogenic hotspot. Other glycosylation sites on EGFR have been implicated in ligand binding, dimerization, and phosphorylation of downstream targets. Molecular dynamic simulations suggested that N361 may be important for dimerization or ligand binding. However, the functional relevance of how glycosylation of EGFR at N361 impacts EGFR protein and cellular behaviors remains unclear. To determine the functional relevance of glycosylation at N361, we created glycosylation-defective EGFR N361A mutant, with or without an additional oncogenic EGFR L858R mutant. We expressed these constructs in MCF10A and 293T cells, which do not have pre-existing activation of this pathway. Immunofluorescence and flow cytometry showed that the mutants were each well expressed at the cell membrane. Proximity ligation assays measuring dimerization of EGFR in cells showed that EGFR N361A greatly increased dimerization relative to wildtype controls. Loss of glycosylation of EGFR N361 impacted cell viability when grown in dose courses of its high affinity ligand EGF or lower affinity ligand amphiregulin. Furthermore, EGFR N361A desensitized cells expressing the oncogenic EGFR L858R to inhibitors targeting the extracellular domain, possibly because the mutation alters the antibody binding interface. N361A sensitized EGFR L858R to inhibitors targeting the cytoplasmic domain of EGFR. These findings help us understand the intricate relationship between EGFR and N-glycosylation, reinforcing the critical functional relevance of post-translational modifications on oncogenes. Citation Format: Dennis Lam, Ariel N. Liberchuk, Brandon Arroyo, Andrew L. Wolfe. Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4365.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Carbon monoxide poisoning: A problem uniquely suited to a medicinal inorganic chemistry solution

Carbon monoxide poisoning is one of the most common forms of poisoning in the world. Although the primary mode of treatment, oxygen therapy, is highly effective in many cases, there are instances in which it is inadequate or inappropriate. Whereas oxygen therapy relies on high levels of a low-affinity ligand (O2) to displace a high-affinity ligand (CO) from metalloproteins, an antidote strategy relies on introducing a molecule with a higher affinity for CO than native proteins (Kantidote,CO > Kprotein,CO). Based on the fundamental chemistry of CO, such an antidote is most likely required to be an inorganic compound featuring an electron-rich transition metal. A review is provided of the protein-, supramolecular complex-, and small molecule-based CO poisoning antidote platforms that are currently under investigation.

Extending the Affinity Range of Weak Affinity Chromatography for the Identification of Weak Ligands Targeting Membrane Proteins.

The identification of weak-affinity ligands targeting membrane proteins is of great interest in Fragment-Based Drug Design (FBDD). Recently, miniaturized weak affinity chromatography (WAC) has been proposed as a valuable tool to study interactions between small ligands and wild-type membrane proteins embedded in so-called nanodisc biomimetic membranes immobilized on GMA-co-EDMA monoliths in situ-synthesized in capillary columns (less than one microliter in volume). In this proof-of-concept study, the achievable affinity range was limited to medium affinity (low micromolar range). The present work investigates different strategies to extend the affinity range towards low affinities, either by increasing the density of membrane proteins on the chromatographic support or by reducing non-specific interactions with the monolith. The combination of the use of a new and more hydrophilic monolithic support (poly(DHPMA-co-MBA)) and a multilayer nanodisc grafting process (up to three layers) allows a significant increase in the membrane protein density by a more than three-fold factor (up to 5.4 pmol cm-1). Such an increase in protein density associated with reduced non-specific interactions makes it possible to extend the range of detectable affinity, as demonstrated by the identification and characterization of affinities of very low-affinity ligands (Kd values of several hundred micromolar) for the adenosine receptor AA2AR used as a model protein, which was not possible before. The affinity was confirmed by competition experiments.

OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids

Abstract Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC50 4.55 nM [3.8;5.3]) compared to GR WT (EC50 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023

Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension

RationalePulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. MethodsTo test this hypothesis, we exposed global (Gpr75−/−) and endothelial cell (EC) GPR75 knockout (EC-Gpr75−/−) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. ResultsChronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75−/− or EC-Gpr75−/− mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75−/−, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. ConclusionThese results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

Transcriptomic signatures reveal a shift towards an anti-inflammatory gene expression profile but also the induction of type I and type II interferon signaling networks through aryl hydrocarbon receptor activation in murine macrophages.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a broad range of target genes involved in the xenobiotic response, cell cycle control and circadian rhythm. AhR is constitutively expressed in macrophages (Mϕ), acting as key regulator of cytokine production. While proinflammatory cytokines, i.e., IL-1β, IL-6, IL-12, are suppressed through AhR activation, anti-inflammatory IL-10 is induced. However, the underlying mechanisms of those effects and the importance of the specific ligand structure are not yet completely understood. Therefore, we have compared the global gene expression pattern in activated murine bone marrow-derived macrophages (BMMs) subsequently to exposure with either benzo[a]pyrene (BaP) or indole-3-carbinol (I3C), representing high-affinity vs. low-affinity AhR ligands, respectively, by means of mRNA sequencing. AhR dependency of observed effects was proved using BMMs from AhR-knockout (Ahr-/-) mice. In total, more than 1,000 differentially expressed genes (DEGs) could be mapped, covering a plethora of AhR-modulated effects on basal cellular processes, i.e., transcription and translation, but also immune functions, i.e., antigen presentation, cytokine production, and phagocytosis. Among DEGs were genes that are already known to be regulated by AhR, i.e., Irf1, Ido2, and Cd84. However, we identified DEGs not yet described to be AhR-regulated in Mϕ so far, i.e., Slpi, Il12rb1, and Il21r. All six genes likely contribute to shifting the Mϕ phenotype from proinflammatory to anti-inflammatory. The majority of DEGs induced through BaP were not affected through I3C exposure, probably due to higher AhR affinity of BaP in comparison to I3C. Mapping of known aryl hydrocarbon response element (AHRE) sequence motifs in identified DEGs revealed more than 200 genes not possessing any AHRE, and therefore being not eligible for canonical regulation. Bioinformatic approaches modeled a central role of type I and type II interferons in the regulation of those genes. Additionally, RT-qPCR and ELISA confirmed a AhR-dependent expressional induction and AhR-dependent secretion of IFN-γ in response to BaP exposure, suggesting an auto- or paracrine activation pathway of Mϕ.

The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.

THEMIS increases TCR signaling in CD4+CD8+ thymocytes by inhibiting the activity of the tyrosine phosphatase SHP1.

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

Effect of Interleukin-2 Inducible Kinase (ITK) on the Temporal Dynamics of TCR Signaling in CD8 T cells

Abstract Antigen affinity has been linked to the rate of downstream TCR activation and the activation of transcription factors such as NFAT and NF-kb. Interleukin-2 inducible T cell kinase (ITK) plays a key role in the differential rate and magnitude of TCR-dependent transcription factor activation, especially when T cells are stimulated with lower affinity ligands. The Nr4a3-Tocky Timer mouse model carries a construct for a fluorescent protein controlled by the Nr4a3 promoter. Nr4a3 is highly expressed following TCR activation and thus is a good metric for TCR signaling. When first produced, the Tocky protein fluoresces “blue,” at peak wavelength 466nm and then, after ~7hrs, degrades to a “red” fluorescence at peak wavelength 583nm. Splenocytes were harvested from OT-I RAG1KO mice crossed to Nr4a3-Tocky mice and stimulated using varying affinities and doses of ova peptide over 7 days in vitro. Cells were exposed to the ITK inhibitor, PRN694, to observe the effect of ITK on the rate of expression of Tocky fluorescence along with activation surface markers CD69, CD25, CD44, and CD122. With ITK inhibition, CD8 T cells expressed the “Blue” protein slower and in fewer cells compared to OT-I cells stimulated without PRN694 at 4hrs. At 48hrs after stimulation, sustained signaling was observed in control cells, whereas a subset of ITK-inhibited cells showed evidence of halted TCR-induced Tocky transcription. This decrease in the longevity of TCR signaling was more evident when lower peptide doses or lower affinity peptides were used for stimulation. These results suggest that ITK plays a role in amplifying the initial TCR signal, and is also required for sustained signaling after TCR activation in CD8 T cells, resulting in the tuning of early gene expression. Supported by grants from the NIH (Dissecting the pathways controlling tunable responses to TCR signaling: 5R01AI132419-06)

PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion

PSGL-1 (P-selectin glycoprotein-1) is a Tcell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the Tcell receptor (TCR) to attenuate activation of mouse and human CD8+ Tcells and drive terminal Tcell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8+ Tcells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating Tcells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8+ Tcell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails Tcell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands.

Membrane proteins may respond to a variety of ligands under an applied external stimulus. These ligands include small low-affinity molecules that account for functional effects in the mM range. Understanding the modulation of protein function by low-affinity ligands requires characterizing their atomic-level interactions under dilution, challenging the current resolution of theoretical and experimental routines. Part of the problem derives from the fact that small low-affinity ligands may interact with multiple sites of a membrane protein in a highly degenerate manner to a degree that it is better conceived as a partition phenomenon, hard to track at the molecular interface of the protein. Looking for new developments in the field, we rely on the classic two-state Boltzmann model to devise a novel theoretical description of the allosteric modulation mechanism of membrane proteins in the presence of small low-affinity ligands and external stimuli. Free energy stability of the partition process and its energetic influence on the protein coupling with the external stimulus are quantified. The outcome is a simple formulation that allows the description of the equilibrium shifts of the protein in terms of the grand-canonical partition function of the ligand at dilute concentrations. The model's predictions of the spatial distribution and response probability shift across a variety of ligand concentrations, and thermodynamic conjugates can be directly compared to macroscopic measurements, making it especially useful to interpret experimental data at the atomic level. Illustration and discussion of the theory is shown in the context of general anesthetics and voltage-gated channels for which structural data are available.

RTK-integrin interactions universally modulate αVβ3 integrin-mediated cell adhesion.

Cells sense and respond to physical forces from the local environment, and aberrations in this bi-directional exchange are implicated in disease. Integrins and receptor tyrosine kinases (RTKs) display robust synergy, with integrins establishing the physical link between the microenvironment and cytoskeleton and RTKs regulating growth, proliferation, and survival. Previously, we found ligand-activated EGFR can tune integrin mechanics, facilitating cell attachment and spreading by influencing focal adhesion (FA) growth and maturation in Cos-7 cells. However, the universality across different RTK family members and cell types and the underlying mechanism remain unknown. To investigate this, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified mechanical outcomes due to RTK activation in three different cell lines belonging to the fibroblast, epithelial and endothelial lineages. Irrespective of the RTK (EGFR, FGFR, or VEGFR) involved, activation with the corresponding ligand increased integrin-based mechanical force generation and consequently enhanced cell spreading, adhesion formation and maturity. Different RTK family members display functional redundancy in terms of the regulated downstream mechanical outcomes and biased agonism to enhance associated changes in cell mechanics. Since multiple ligands can activate RTKs with distinct signaling outcomes, we investigated the influence of different ligands on cell mechanics. Ligand affinity inversely correlated with cell mechanical changes where low-affinity ligands increased integrin activation and spreading via sustained RTK signaling. Finally, the concentration of the available ligand directly impacts the observed changes in mechanical outcomes. Together, different ligand availability provides an additional layer of control to ‘fine-tune’ cell mechanical outcomes. Our results reveal RTKs as universal mechano-organizers which can synergistically function with integrins as joint-signaling rheostats to modulate adhesion and cell spreading.

Rosetta's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery.

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here, we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF (UniProtKB Q9X0C6). We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability using RosettaLigand and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand's ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 μM. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve of the receiver operating characteristics of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands

Time required for commitment to T cell proliferation depends on TCR affinity and cytokine response.

T cell activation and effector functions are determined by the affinity of the interaction between T cell receptor (TCR) and its antigenic peptide MHC (pMHC) ligand. A better understanding of the quantitative aspects of TCR-pMHC affinity-dependent T cell activation is critical for the development of new immunotherapeutic strategies. However, the role of TCR-pMHC affinity in regulating the kinetics of CD8+ T cell commitment to proliferation and differentiation is unknown. Here, we show that the stronger the TCR-pMHC affinity, the shorter the time of T cell-APC co-culture required to commit CD8+ T cells to proliferation. The time threshold for T cell cytokine production is much lower than that for cell proliferation. There is a strong correlation between affinity-dependent differences in AKT phosphorylation and T cell proliferation. The cytokine IL-15 increases the poor proliferation of T cells stimulated with low affinity pMHC, suggesting that pro-inflammatory cytokines can override the affinity-dependent features of T cell proliferation.