Cryo-EM studies indicate that full-length human TSH receptor (TSHR) is a monomer consisting of a leucine-rich repeat (LRR) domain (LRD), hinge region (HR) and transmembrane domain (TMD). Receptor autoantibodies (TRAb) bind to the extracellular domain (LRD plus HR). In the cell membrane, TSHR transitions between inactive and active states, involving rotation of the LRD about the HR, with the active state LRD further from the membrane. Cryo-EM structures of the TSHR in complex with different human monoclonal autoantibodies provide a detailed understanding of how they interact with the receptor. Stimulating monoclonal autoantibody M22™ binds to the entire LRD (LRR 1–11) concave surface and cannot interact with the inactive state receptor, as this would result in a clash between antibody and membrane. No clash occurs when M22™ binds the active state, and once bound, M22™ holds the receptor in the active state, resulting in prolonged activation. K1-18™, a different stimulating monoclonal autoantibody, interacts with LRR 1–11 in a similar way to M22™. Blocking type monoclonal autoantibody K1-70™ only interacts with LRR 1–7, positioning itself clear of the cell membrane when interacting with inactive or active receptor states. Once bound, binding of TSH and other TRAb is prevented. Monoclonal autoantibody 5C9™ interacts differently, binding the inactive conformation from LRR 3 to the HR. Its heavy chain CDR3 interacts with the HR, locking the receptor in the inactive state, causing inhibition of constitutive activity, activating mutation activity and blocking receptor binding by TSH and TRAb. Elucidation of the structure of the TSHR and mechanisms of its activation and inactivation by TRAb is of great importance for the development of new TSHR-specific drugs.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the hTPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92 and 3.4 Å for the 2G4 complex and 4F5 complex, respectively) show TPO as a monomer with four domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein (CCP)-like domain and the epidermal growth factor-like domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B mainly via different residues. However, the epitopes of the two antibodies contain three shared TPO residues. This is the first high-resolution structure of TPO to be reported and it should help guide the development of new inhibitors of TPO enzyme activity for therapeutic applications.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

Supplementary Methods from Targeting LMO2 with a Peptide Aptamer Establishes a Necessary Function in Overt T-Cell Neoplasia

Supplementary Figure 3 from Targeting LMO2 with a Peptide Aptamer Establishes a Necessary Function in Overt T-Cell Neoplasia

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

Checkpoint blockade immunotherapy (CBI) targeting the PD-1 and CTLA-4 pathways is revolutionizing oncology. However, objective response rates for single agent CBI in solid malignancies remain low and combinatorial strategies are required to improve outcomes. Our group has pioneered the use of radiation (RT) and stereotactic body radiation therapy (SBRT) in combination with anti-PD-1 CBI. We previously demonstrated in tumor models that RT+ CBI can enhance antigen specific immune responses and improve local and systemic tumor control. Nevertheless, the clinical benefit of SBRT+CBI remains unclear. Our primary objective was to conduct a clinical trial to test the hypothesis that SBRT + CBI increases objective response rates (ORR) compared to CBI alone. Here we present an interim analysis. We designed and opened an investigator initiated Phase II study (ClinicalTrials.gov NCT02843165) which randomizes patients (1:1) with advanced metastatic disease to receive investigators choice of anti-PD-1/PD-L1 CBI versus anti-PD-1/PD-L1 CBI combined with concurrent SBRT. SBRT was started 1-21 days after C1D1 of CBI and was prescribed at 9.5Gy x 3 fractions delivered to 1-3 non-CNS tumor foci consecutively or every other day. The study is powered to detect a 33% relative improvement in ORR with a total anticipated enrollment of 146. Safety and Toxicity was analyzed prospectively for all patients using CTCAE 4.0. ORR are measured from unirradiated tumors using RECIST 1.1 criteria. Three research blood draws are obtained to analyze immune correlates. We performed an interim analysis of safety and toxicity among all patients and between treatment groups. We have enrolled a total of 35 patients at this time. There were a total of 126 CTCAE toxicity events observed with 67 (53.2%) Low grade (CTCAE 1-2), and 59 (46.8%) High grade (CTCAE 3-5) events with a mean followup of 5.1 months. The most common toxicities were fatigue, nausea, pain, and rash. The most common SBRT treatment sites were lung, liver, adrenal gland, and soft tissue. Experimental treatment attributions are as follows: 18 events (14.3%) Unrelated, 44 events (34.9%) Unlikely, 48 events (38.1%) Possible, and 13 events (10.3%) Probable or Definite. There were 7 (5.5%) high grade events attributed as probably or definitely related to study: one grade 4 fatigue, one grade 3 weight loss, one grade 3 rash, and two grade 3 electrolyte abnormalities, and two grade 3 acute kidney injury. When comparing between CBI and CBI+SBRT treatment groups there was no significant difference in any grade of toxicity at this time. Our interim analysis suggests that SBRT to lung, liver, adrenal and soft tissues prescribed at 9.5Gy x 3 fractions combined with concurrent anti-PD-1/PD-L1 CBI appears to be safe and well tolerated in patients with advanced metastatic disease. Continued analysis of safety and toxicity, ORR, and immune correlates between randomized treatment groups is ongoing.

Purpose The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves' disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD-M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown.Methods We have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available.Results Most conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs.Conclusions Insight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves' disease.

We have studied glycosylation patterns in glycoprotein hormones (GPHs) and glycoprotein hormone receptor (GPHR) extracellular domains (ECD) from different species to identify areas not glycosylated that could be involved in intermolecular or intramolecular interactions. Comparative models of the structure of the TSHR ECD in complex with TSH and in complex with TSHR autoantibodies (M22, stimulating and K1-70, blocking) were obtained based on the crystal structures of the FSH-FSHR ECD, M22-TSHR leucine-rich repeat domain (LRD) and K1-70-TSHR LRD complexes. The glycosylation sites of the GPHRs and GPHs from all species studied were mapped on the model of the human TSH TSHR ECD complex. The areas on the surfaces of GPHs that are known to interact with their receptors are not glycosylated and two areas free from glycosylation, not involved in currently known interactions, have been identified. The concave faces of GPHRs leucine-rich repeats 3-7 are free from glycosylation, consistent with known interactions with the hormones. In addition, four other non-glycosylated areas have been identified, two located on the receptors' convex surfaces, one in the long loop of the hinge regions and one at the C-terminus of the extracellular domains. Experimental evidence suggests that the non-glycosylated areas identified on the hormones and receptors are likely to be involved in forming intramolecular or intermolecular interactions.

The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1-70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively. K1-70 has important potential in the treatment of the hyperthyroidism of Graves' disease and Graves' ophthalmopathy. Small molecule TSHR antagonists described to date do not appear to have the potency and/or specificity shown by K1-70. New models of the TSHR ECD in complex with various ligands have been built. These models suggest that initial binding of TSH to the TSHR causes a conformational change in the hormone. This opens a positively charged pocket in receptor-bound TSH which attracts the negatively charged sulphated tyrosine 385 on the hinge region of the receptor. The ensuing movement of the receptor's hinge region may then cause activation. Similar activation mechanisms seem to take place in the case of FSH and the FSHR and LH and the LHR. However, stimulating TSHR autoantibodies do not appear to activate the TSHR in the same way as TSH.

eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes.We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins.eIF4E1b exhibited only very weak interactions with m 7 GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T.Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling.The predicted structure of eIF4E1b maintains the α + β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop.However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N 7 of guanine.Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues.Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N 7 position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core.In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.

Binding of a new thyroid-stimulating human monoclonal autoantibody (MAb) K1-18 to the TSH receptor (TSHR) leucine-rich domain (LRD) was predicted using charge-charge interaction mapping based on unique complementarities between the TSHR in interactions with the thyroid-stimulating human MAb M22 or the thyroid-blocking human MAb K1-70. The interactions of K1-18 with the TSHR LRD were compared with the interactions in the crystal structures of the M22-TSHR LRD and K1-70-TSHR LRD complexes. Furthermore, the predicted position of K1-18 on the TSHR was validated by the effects of TSHR mutations on the stimulating activity of K1-18. A similar approach was adopted for predicting binding of a mouse thyroid-blocking MAb RSR-B2 to the TSHR. K1-18 is predicted to bind to the TSHR LRD in a similar way as TSH and M22. The binding analysis suggests that K1-18 light chain (LC) mimics binding of the TSH-α chain and the heavy chain (HC) mimics binding of the TSH-β chain. By contrast, M22 HC mimics the interactions of TSH-α while M22 LC mimics TSH-β in interactions with the TSHR. The observed interactions in the M22-TSHR LRD and K1-70-TSHR LRD complexes (crystal structures) with TSH-TSHR LRD (comparative model) and K1-18-TSHR LRD (predictive binding) suggest that K1-18 and M22 interactions with the receptor may reflect interaction of thyroid-stimulating autoantibodies in general. Furthermore, K1-70 and RSR-B2 interactions with the TSHR LRD may reflect binding of TSHR-blocking autoantibodies in general. Interactions involving the C-terminal part of the TSHR LRD may be important for receptor activation by autoantibodies.

Summary The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes . PrfA‐dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand‐induced allostery. Here, we report the identification of a solvent‐accessible cavity within the PrfA N‐terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide‐anchoring motif and has its entrance on the opposite side of the β‐barrel. Site‐directed mutations in this pocket impaired intracellular PrfA‐dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation‐deficient PrfA mutations. Neither PrfA allo substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell‐to‐cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues.

Intracellular trafficking and protein sorting are mediated by various protein complexes, with the retromer complex being primarily involved in retrograde traffic from the endosome or lysosome to the Golgi complex. Here, comparative genomics, cell biology and phylogenetics were used to probe the early evolution of retromer and its function. Retromer subunits Vps26, Vps29 and Vps35 are near universal, and, by inference, the complex was an ancient feature of eukaryotic cells. Surprisingly, we found DSCR3, a Vps26 paralogue in humans associated with Down's syndrome, in at least four eukaryotic supergroups, implying a more ancient origin than previously suspected. By contrast, retromer cargo proteins showed considerable interlineage variability, with lineage-specific and broadly conserved examples found. Vps10 trafficking probably represents an ancestral role for the complex. Vps5, the BAR-domain-containing membrane-deformation subunit, was found in diverse eukaryotes, including in the divergent eukaryote Trypanosoma brucei, where it is the first example of a BAR-domain protein. To determine functional conservation, an initial characterisation of retromer was performed in T. brucei; the endosomal localisation and its role in endosomal targeting are conserved. Therefore retromer is identified as a further feature of the sophisticated intracellular trafficking machinery of the last eukaryotic common ancestor, with BAR domains representing a possible third independent mechanism of membrane-deformation arising in early eukaryotes.

A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1.9 Å resolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å(2)) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Å resolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with 'lock and key' binding. The binding arrangements in the TSHR260-K1-70 Fab complex are similar to previously observed for the TSHR260-M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260-K1-70 and TSHR260-M22 complexes show a root mean square deviation on all C(α) atoms of only 0.51 Å. These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

Autoantibodies in autoimmune thyroid disease (AITD) bind to the TSH receptor (TSHR) and can act as either agonists, mimicking the biological activity of TSH, or as antagonists inhibiting the action of TSH. Furthermore, some antibodies with antagonist activity can also inhibit the constitutive activity of the TSHR, that is, act as inverse agonists. The production of animal TSHR monoclonal antibodies (MAbs) with the characteristics of patient autoantibodies and the isolation of human autoantibodies from patients with AITD has allowed us to analyze the interactions of these antibodies with the TSHR at the molecular level.In the case of animal MAbs, advances such as DNA immunization allowed the production of the first MAbs which showed the characteristics of human TSHR autoantibodies (TRAbs). Mouse MAbs (TSMAbs 1-3) and a hamster MAb (MS-1) were obtained that acted as TSHR agonists with the ability to stimulate cyclic AMP production in CHO cells expressing the TSHR. In addition, a mouse TSHR MAb (MAb-B2) that had the ability to act as an antagonist of TRAbs and TSH was isolated and characterized. Also, a mouse TSHR MAb that showed TSH antagonist and TSHR inverse agonist activity (CS-17) was described.Furthermore, a panel of human TRAbs has been obtained from the peripheral blood lymphocytes of patients with AITD and extensively characterized. These MAbs have all the characteristics of TRAbs and are active at ng/mL levels. To date, two human MAbs with TSHR agonist activity (M22 and K1-18), one human MAb with TSHR antagonist activity (K1-70) and one human MAb (5C9) with both TSHR antagonist and TSHR inverse agonist activity have been isolated.Early experiments showed that the binding sites for TSH and for TRAbs with thyroid stimulating or blocking activities were located on the extracellular domain of the TSHR. Extensive studies using TSHRs with single amino acid mutations identified TSHR residues that were important for binding and biological activity of TSHR MAbs (human and animal) and TSH. The structures of several TSHR MAb Fab fragments were solved by X-ray crystallography and provided details of the topography of the antigen binding sites of antibodies with either agonist or antagonist activity. Furthermore stable complexes of the leucine-rich repeat domain (LRD) of the TSHR with a human MAb (M22) with agonist activity and with a human MAb (K1-70) with antagonist activity have been produced and their structures solved by X-ray crystallography at 2.55 and 1.9 angstrom resolution, respectively. Together these experiments have given detailed insights into the interactions of antibodies with different biological activities (agonist, antagonist, and inverse agonist) with the TSHR.Although the nature of ligand binding to the TSHR is now understood in some detail, it is far from clear how these initial interactions lead to functional effects on activation or inactivation of the receptor.

Background: The Y chromosome of mammals is particularly prone to accumulate genes related to male fertility. However, the high rate of molecular evolution on this chromosome predicts reduced power to the across-species comparative approach in identifying male-specific genes that are essential for sperm production in humans. We performed a comprehensive analysis of expression of Y-linked transcripts and their X homologues in several human tissues, and in biopsies of infertile patients, in an attempt to identify new testis-specific genes involved in human spermatogenesis.Results: We present evidence that one of the primate-specific Y-linked ribosomal protein genes, RPS4Y2, has restricted expression in testis and prostate, in contrast with its X-linked homologue, which is ubiquitously expressed. Moreover, we have determined by highly specific quantitative real time PCR that RPS4Y2 is more highly expressed in testis biopsies containing germ cells. The in silico analysis of the promoter region of RPS4Y2 revealed several differences relative to RPS4Y1, the more widely expressed paralogue from which Y2 has originated through duplication. Finally, through comparative modelling we obtained the three dimensional models of the human S4 proteins, revealing a conserved structure. Interestingly, RPS4Y2 shows different inter-domain contacts and the potential to establish specific interactions.Conclusions: These results suggest that one of the Y-linked copies of the ribosomal protein S4 is preferentially expressed during spermatogenesis and might be important for germ cell development. Even though RPS4Y2 has accumulated several amino acid changes following its duplication from RPS4Y1, approximately 35 million years ago, the evolution of the Y-encoded RPS4 proteins is structurally constrained. However, the exclusive expression pattern of RPS4Y2 and the novelties acquired at the C-terminus of the protein may indicate some degree of functional specialisation of this protein in spermatogenesis.