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Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Collision cross-sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift-tube ion-mobility mass spectrometry. Intact antibodies and Fc-hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

RATIONALE Non-covalent mass spectrometry (MS) offers considerable potential for protein-ligand screening in drug discovery programmes. However, there are some limitations with the time-of-flight (TOF) instrumentation typically employed that restrict the application of non-covalent MS in industrial laboratories. METHODS An Exactive Plus EMR mass spectrometer was investigated for its ability to characterise non-covalent protein–small molecule interactions. Nano-electrospray ionisation (nanoESI) infusion was achieved with a TriVersa NanoMate. The transport multipole and ion lens voltages, dissociation energies and pressure in the Orbitrap™ were optimised. Native MS was performed, with ligand titrations to judge retention of protein-ligand interactions, serial dilutions of native proteins as an indication of sensitivity, and a heterogeneous protein analysed for spectral resolution. RESULTS Interactions between native proteins and ligands are preserved during analysis on the Exactive Plus EMR, with the binding affinities determined in good agreement with expected values. High spectral resolution allows baseline separation of adduct ions, which should improve the accuracy and limit of detection for measuring ligand interactions. Data are also presented showing baseline resolution of glycoforms of a highly glycosylated protein, allowing binding of a fragment molecule to be detected. CONCLUSIONS The high sensitivity and spectral resolution achievable with the Orbitrap technology confer significant advantages over TOF mass spectrometers, and offer a solution to current limitations regarding throughput, data analysis and sample requirements. A further benefit of improved spectral resolution is the possibility of using heterogeneous protein samples such as glycoproteins for fragment screening. This would significantly expand the scope of applicability of non-covalent MS in the pharmaceutical and other industries. Copyright © 2014 John Wiley & Sons, Ltd.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

Summary MtATP-phosphoribosyltransferase (MtATP-PRT) is an enzyme catalyzing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, and proposed to be regulated via an allosteric mechanism. Native mass spectrometry (MS) reveals MtATP-PRT to exist as a hexamer. Conformational changes induced by L-histidine binding and the influence of buffer pH are determined with ion mobility MS, hydrogen deuterium exchange (HDX) MS, and analytical ultracentrifugation. The experimental collision cross-section (DTCCSHe) decreases from 76.6 to 73.5 nm2 upon ligand binding at pH 6.8, which correlates to the decrease in CCS calculated from crystal structures. No such changes in conformation were found at pH 9.0. Further detail on the regions that exhibit conformational change on L-histidine binding is obtained with HDX-MS experiments. On incubation with L-histidine, rapid changes are observed within domain III, and around the active site at longer times, indicating an allosteric effect., Graphical Abstract, Highlights • Hybrid MS approaches map global and local conformational changes in MtATP- PRT • IM-MS shows hexameric MtATP-PRT to undergo conformational change on L-histidine binding • HDX-MS maps conformational changes to regions close to and remote from the active site, MtATP-phosphoribosyltransferase (MtATP-PRT) catalyzes the first step in Mycobacterium tuberculosis L-histidine biosynthesis. Pacholarz et al. use mass spectrometry approaches to show that MtATP-PRT is a hexamer, and use measurements at different pH values to demonstrate that L-histidine allosteric effect does not alter the oligomeric state of the enzyme.

Non-covalent mass spectrometry (MS) offers considerable potential for protein-ligand screening in drug discovery programmes. However, there are some limitations with the time-of-flight (TOF) instrumentation typically employed that restrict the application of non-covalent MS in industrial laboratories.An Exactive Plus EMR mass spectrometer was investigated for its ability to characterise non-covalent protein-small molecule interactions. Nano-electrospray ionisation (nanoESI) infusion was achieved with a TriVersa NanoMate. The transport multipole and ion lens voltages, dissociation energies and pressure in the Orbitrap™ were optimised. Native MS was performed, with ligand titrations to judge retention of protein-ligand interactions, serial dilutions of native proteins as an indication of sensitivity, and a heterogeneous protein analysed for spectral resolution.Interactions between native proteins and ligands are preserved during analysis on the Exactive Plus EMR, with the binding affinities determined in good agreement with expected values. High spectral resolution allows baseline separation of adduct ions, which should improve the accuracy and limit of detection for measuring ligand interactions. Data are also presented showing baseline resolution of glycoforms of a highly glycosylated protein, allowing binding of a fragment molecule to be detected.The high sensitivity and spectral resolution achievable with the Orbitrap technology confer significant advantages over TOF mass spectrometers, and offer a solution to current limitations regarding throughput, data analysis and sample requirements. A further benefit of improved spectral resolution is the possibility of using heterogeneous protein samples such as glycoproteins for fragment screening. This would significantly expand the scope of applicability of non-covalent MS in the pharmaceutical and other industries.