The full-length TSH receptor is stabilized by TSH ligand.

The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the "hinge region", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.

Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

Functional Water Channels Within the TSH Receptor: A New Paradigm for TSH Action With Disease Implications.

The thyroid-stimulating hormone receptor (TSHR) transmembrane domain (TMD) is found in the plasma membrane and consists of lipids and water molecules. To understand the role of TSHR-associated water molecules, we used molecular dynamic simulations of the TMD and identified a network of putative receptor-associated transmembrane water channels. This result was confirmed with extended simulations of the full-length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself, we found that significantly more water traversed via the TSHR than via the lipid bilayer, which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique, we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport, and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport, illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across 2 activation motifs within the TSHR TMD, suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable, this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed, thus affecting normal cellular function.

Modeling TSH Receptor Dimerization at the Transmembrane Domain.

Biophysical studies have established that the thyrotropin (TSH) receptor (TSHR) undergoes posttranslational modifications including dimerization. Following our earlier simulation of a TSHR-transmembrane domain (TMD) monomer (called TSHR-TMD-TRIO) we have now proceeded with a molecular dynamics simulation (MD) of TSHR-TMD dimerization using this improved membrane-embedded model. The starting structure was the TMD protein with all extracellular and intracellular loops and internal waters, which was placed in the relative orientation of the model originally generated with Brownian dynamics. Furthermore, this model was embedded in a DPPC lipid bilayer further solvated with water and added salt. Data from the MD simulation studies showed that the dimeric subunits stayed in the same relative orientation and distance during the 1000 ns of study. Comparison of representative conformations of the individual monomers when dimerized with the conformations from the monomer simulation showed subtle differences as represented by the backbone root mean square deviations. Differences in the conformations of the ligand-binding sites, suggesting variable affinities for these "hot spots," were also revealed by comparing the docking scores of 46 small-molecule ligands that included known TSHR agonists and antagonists as well as their derivatives. These data add further insight into the tendency of the TSHR-TMD to form dimeric and oligomeric structures and show that the differing conformations influence small-molecule binding sites within the TMD.

Computational model of the full-length TSH receptor.

(GPCR)The receptor for TSH receptor (TSHR), a G protein coupled receptor (GPCR), is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have run a 1000 ns molecular dynamic simulation on a model of the entire TSHR generated by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane and solvated it with water and counterions. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linker region (LR), known more commonly as the 'hinge region,' showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also seen to be variable. These data suggest that this LR is an intrinsically disordered protein. Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the LR and TSH ligand and (b) the association of the LR and the TSH ligand reduces the structural fluctuations in the LR. This full-length model illustrates the importance of the LR in responding to ligand binding and lays the foundation for studies of pathologic TSHR autoantibodies complexed with the TSHR to give further insight into their interaction with the flexible LR.

Brief Report - Monoclonal Antibodies Illustrate the Difficulties in Measuring Blocking TSH Receptor Antibodies.

TSH receptor (TSHR) antibodies are the cause of Graves' disease and may also be found in patients with Hashimoto's thyroiditis. They come in at least three varieties: thyroid stimulating, thyroid blocking and neutral. The measurement of TSH receptor antibodies in Graves' disease and Hashimoto's thyroiditis is a common clinical activity and can be useful in diagnosis and prognosis. We show that it is not possible to detect the blocking variety of TSHR antibody in patients with Graves' disease because the stimulating antibody may overwhelm the measurement of blocking in the bioassays available for their measurement and may blind the valid interpretation of the results. To help explain this in more detail we show a series of studies with monoclonal TSHR antibodies which support this conclusion.

Tools for Characterizing Proteins: Circular Variance, Mutual Proximity, Chameleon Sequences, and Subsequence Propensities.

For the characterization of various aspects of protein structures, four useful concepts are discussed: chameleon sequences, circular variance, mutual proximity, and a subsequence-based foldability score. These concepts were used in estimating foldability of globular, intrinsically disordered and fold-switching proteins, properties of protein-protein interfaces, quantifying sphericity, helping to improve protein-protein docking scores, and estimating the effect of mutations on stability. A conjecture about the Achilles' heel of proteins is presented as well.

An NR2F1-specific agonist suppresses metastasis by inducing cancer cell dormancy.

We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 agonists to induce dormancy as a therapeutic strategy to prevent metastasis.

The Biological Significance of Targeting Acetylation-Mediated Gene Regulation for Designing New Mechanistic Tools and Potential Therapeutics.

The molecular interplay between nucleosomal packaging and the chromatin landscape regulates the transcriptional programming and biological outcomes of downstream genes. An array of epigenetic modifications plays a pivotal role in shaping the chromatin architecture, which controls DNA access to the transcriptional machinery. Acetylation of the amino acid lysine is a widespread epigenetic modification that serves as a marker for gene activation, which intertwines the maintenance of cellular homeostasis and the regulation of signaling during stress. The biochemical horizon of acetylation ranges from orchestrating the stability and cellular localization of proteins that engage in the cell cycle to DNA repair and metabolism. Furthermore, lysine acetyltransferases (KATs) modulate the functions of transcription factors that govern cellular response to microbial infections, genotoxic stress, and inflammation. Due to their central role in many biological processes, mutations in KATs cause developmental and intellectual challenges and metabolic disorders. Despite the availability of tools for detecting acetylation, the mechanistic knowledge of acetylation-mediated cellular processes remains limited. This review aims to integrate molecular and structural bases of KAT functions, which would help design highly selective tools for understanding the biology of KATs toward developing new disease treatments.

Implications of an Improved Model of the TSH Receptor Transmembrane Domain (TSHR-TMD-TRIO).

The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model, which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD-TRIO since 3 representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The 3 models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules showing significant preference for one or other of the structures.

Observation of quantum signature in rivastigmine chemical bond break-up and quantum energetics, spectral studies of anti-Alzheimer inhibitors.

Semi-empirical calculations on the torsion potential for dihedral angle of rivastigmine linking NAP and Carbamyle moieties consistently show regions of several discontinuities and a cusp indicating molecular instability and eventual break-up of rivastigmine observed in the X-ray structure. The phenomena can be explained both by definition of large classical force or quantum nature of chemical bond break-up. Also, to better understand the molecular properties and quantum energetics of the inhibitor molecules, we have performed several ab initio based calculations on all four inhibitors at equilibrium geometry, in ground state and gas phase using the density functional theory level wB97X/6-31G* and HF/6-31G*. A number of properties like computational vibrational (IR), Raman and nuclear magnetic resonance (NMR) spectra as well as HOMO and LUMO orbital energies at optimized geometries have been computed by SPARTAN16 and Gaussian16 utilities. Also, the thermodynamic and QSAR properties of the inhibitors have been assessed and compared by a number of different semi-quantum, Hartree-Fock and density functional methods. The theoretical NMR and IR spectra have been benchmarked against experimental spectrum to compare and assess suitability of the computational methodologies and basis set levels for the calculations.Communicated by Ramaswamy H. Sarma.

Foldability and chameleon propensity of fold-switching protein sequences.

It has been shown recently by Porter & Looger that a significant number of proteins exist that can form more than one stable fold. This note examines the sequences of these fold-switching proteins by (a) calculating their foldability scores recently introduced by the present author and (b) comparing the propensity of chameleon sequences in fold switchers and in non fold switchers. It has been found that the average foldability score of the fold switchers indicates weaker foldability. As for the propensity of chameleon sequences of length 5 to 7 it was found, somewhat surprisingly, that there is only a very small difference between the fold switchers and the non fold switchers. Furthermore, when looking at amino acid propensities, for several amino acids there was even an opposing trend in the deviation of their propensities from the overall amino acid propensities.

Retro-inverso D-peptides as a novel targeted immunotherapy for Type 1 diabetes.

Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-d-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23-DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.

The Human TSHß Subunit Proteins and Their Binding Sites on the TSH Receptor Using Molecular Dynamics Simulation.

To gain further insight into the binding of the normal and variant human TSHß subunits (TSHß and TSHßv), we modeled the 2 monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) using molecular dynamics simulation Furthermore, analyzed their bioactivity in vitro using recombinant proteins to confirm that such binding was physiologically relevant. Examining the interaction of TSHß and TSHßv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHß and TSHßv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD, indicating the inability of these molecules to bind to the FSH receptors. Furthermore, the modelling suggests that TSHß and TSHßv proteins clasped the concave surface of the leucine rich region of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high-affinity contact sites corresponding to binding models of native TSH. Furthermore, we cloned TSHß and TSHßv proteins using the entire coding ORF and purified the flag-tagged proteins. The expressed TSHß subunit proteins retained bioactivity both in a coculture system as well as with immune-purified proteins. In summary, we showed that such interactions can result in a functional outcome and may exert physiological or pathophysiological effects in immune cells.

A Gq Biased Small Molecule Active at the TSH Receptor.

G protein coupled receptors (GPCRs) can lead to G protein and non-G protein initiated signals. By virtue of its structural property, the TSH receptor (TSHR) has a unique ability to engage different G proteins making it highly amenable to selective signaling. In this study, we describe the identification and characterization of a novel small molecule agonist to the TSHR which induces primary engagement with Gαq/11. To identify allosteric modulators inducing selective signaling of the TSHR we used a transcriptional-based luciferase assay system with CHO-TSHR cells stably expressing response elements (CRE, NFAT, SRF, or SRE) that were capable of measuring signals emanating from the coupling of Gαs , Gαq/11, Gßγ, and Gα12/13, respectively. Using this system, TSH activated Gαs , Gαq/11, and Gα12/13 but not Gßγ. On screening a library of 50K molecules at 0.1,1.0 and 10 µM, we identified a novel Gq/11 agonist (named MSq1) which activated Gq/11 mediated NFAT-luciferase >4 fold above baseline and had an EC50= 8.3 × 10-9 M with only minor induction of Gαs and cAMP. Furthermore, MSq1 is chemically and structurally distinct from any of the previously reported TSHR agonist molecules. Docking studies using a TSHR transmembrane domain (TMD) model indicated that MSq1 had contact points on helices H1, H2, H3, and H7 in the hydrophobic pocket of the TMD and also with the extracellular loops. On co-treatment with TSH, MSq1 suppressed TSH-induced proliferation of thyrocytes in a dose-dependent manner but lacked the intrinsic ability to influence basal thyrocyte proliferation. This unexpected inhibitory property of MSq1 could be blocked in the presence of a PKC inhibitor resulting in derepressing TSH induced protein kinase A (PKA) signals and resulting in the induction of proliferation. Thus, the inhibitory effect of MSq1 on proliferation resided in its capacity to overtly activate protein kinase C (PKC) which in turn suppressed the proliferative signal induced by activation of the predomiant cAMP-PKA pathway of the TSHR. Treatment of rat thyroid cells (FRTL5) with MSq1 did not show any upregulation of gene expression of the key thyroid specific markers such as thyroglobulin(Tg), thyroid peroxidase (Tpo), sodium iodide symporter (Nis), and the TSH receptor (Tshr) further suggesting lack of involvement of MSq1 and Gαq/11 activation with cellular differentation. In summary, we identified and characterized a novel Gαq/11 agonist molecule acting at the TSHR and which showed a marked anti-proliferative ability. Hence, Gq biased activation of the TSHR is capable of ameliorating the proliferative signals from its orthosteric ligand and may offer a therapeutic option for thyroid growth modulation.

Protein Binding Pocket Optimization for Virtual High-Throughput Screening (vHTS) Drug Discovery.

The virtual high-throughput screening (vHTS) approach has been widely used for large database screening to identify potential lead compounds for drug discovery. Due to its high computational demands, docking that allows receptor flexibility has been a challenging problem for virtual screening. Therefore, the selection of protein target conformations is crucial to produce useful vHTS results. Since only a single protein structure is used to screen large databases in most vHTS studies, the main challenge is to reduce false negative rates in selecting compounds for in vitro tests. False negatives are most likely to occur when using apo structures or homology models of protein targets due to the small volume of the binding pocket formed by incorrect side-chain conformations. Even holo protein structures can exhibit high false negative rates due to ligand-induced fit effects, since the shape of the binding pocket highly depends on its bound ligand. To reduce false negative rates and improve success rates for vHTS in drug discovery, we have developed a new Monte Carlo-based approach that optimizes the binding pocket of protein targets. This newly developed Monte Carlo pocket optimization (MCPO) approach was assessed on several datasets showing promising results. The binding pocket optimization approach could be a useful tool for vHTS-based drug discovery, especially in cases when only apo structures or homology models are available.

On predicting foldability of a protein from its sequence.

Several properties of amino acid sequences corresponding to proteins that are known to fold are compared to those of randomly generated sequences and to sequences of intrinsically disordered proteins in order to find properties that distinguish folding sequences from the rest. The properties studied included helix and sheet propensities from secondary structure prediction, adjacency correlations, directionality correlations, as well as propensities of all possible triplets and quadruplets. Small differences between known folded and random sequences were observed for the adjacency and directional correlations, and significant differences were seen on the triplet and especially on the quadruplet propensities. Based on the differences in the adjacency, triplet or quadruplet propensities folding scores were defined and used to test the accuracy of foldability prediction based on these statistics. The best predictions were obtained from the quadruplet propensities.

A Modifying Autoantigen in Graves' Disease.

The TSH receptor (TSHR) is the major autoantigen in Graves' disease (GD). Bioinformatic analyses predict the existence of several human TSHR isoforms from alternative splicing, which can lead to the coexpression of multiple receptor forms. The most abundant of these is TSHRv1.3. In silico modeling of TSHRv1.3 demonstrated the structural integrity of this truncated receptor isoform and its potential binding of TSH. Tissue profiling revealed wide expression of TSHRv1.3, with a predominant presence in thyroid, bone marrow, thymus, and adipose tissue. To gain insight into the role of this v1.3 receptor isoform in thyroid pathophysiology, we cloned the entire open reading frame into a mammalian expression vector. Immunoprecipitation studies demonstrated that both TSHR-stimulating antibody and human TSH could bind v1.3. Furthermore, TSHRv1.3 inhibited the stimulatory effect of TSH and TSHR-Ab MS-1 antibody on TSHR-induced cAMP generation in a dose-dependent manner. To confirm the antigenicity of v1.3, we used a peptide ELISA against two different epitopes. Of 13 GD samples, 11 (84.6%) were positive for a carboxy terminal peptide and 10 (76.9%) were positive with a junction region peptide. To demonstrate that intracellular v1.3 could serve as an autoantigen and modulate disease, we used double-transfected Chinese hamster ovary cells that expressed both green fluorescent protein (GFP)-tagged TSHRv1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR monoclonal antibody and observed the culture supernatant contained v1.3-GFP protein, demonstrating the release of the intracellular receptor variant by this mechanism.

Virtual screening of a natural compound library at orthosteric and allosteric binding sites of the neurotensin receptor.

Molecular dynamics (MD) simulation using the AMBER force field has been performed on the neurotensin (NT) receptor, a class A type G-protein-coupled receptor in its activated conformation co-crystallized with the non-peptide agonists. For structure-based hit molecule identification via natural chemical compound library, orthosteric sites on NT receptor have been mapped by docking using AutoDock4.0 and Vina with the known agonists and antagonists SR48692, SR142948, ML301 and ML314 of the receptor. Furthermore, clustering analysis on the MD trajectories by SIMULAID has been performed to filter receptor conformations for the allosteric binders from the Otava natural compound library. Comparative mappings of contrasting binding region patterns have been done between the crystal structure orthosteric sites as well as the binding regions in the SIMULAID-based cluster center conformations from MD trajectories with the FTmap server using the small organic molecule fragments as the probes. The distinct binding region in the cluster-based conformations in the extracellular region of the receptor has been identified for targeted docking by Otava natural chemical compound library using AutoDock4.0 and Vina docking suites to obtain putative allosteric binders. A group of compounds from the Otava library has been identified as showing high free energy in both AutoDock4.0 and Vina docking suites. Biophysical assessments on the natural compound computational hit molecules will be done to identify lead structures from the hit molecules. Communicated by Ramaswamy H. Sarma.

Inhibitor of CBP Histone Acetyltransferase Downregulates p53 Activation and Facilitates Methylation at Lysine 27 on Histone H3.

Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.