Search

Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.

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.

Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cɛ2 domain pair folded back onto the Cɛ3-Cɛ4 domains, but is the molecule exclusively bent or can the Cɛ2 domains adopt extended conformations and even 'flip' from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, surface plasmon resonance (SPR) and Forster resonance energy transfer (FRET) analyses that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B-cell receptor and as a therapeutic target in allergic disease.

Immunoglobulin E (IgE) antibodies mediate allergic reactions, and their binding to FcaRI is responsible for longterm sensitisation of mast cells and basophils [1]. Disruption of this interaction is a validated strategy for therapeutic intervention in allergic diseases including asthma [2]. IgE is known to display an acutely and asymmetrically bent conformation in the Fc region through which it binds to FcaRI; this bend becomes even more acute upon receptor engagement, as shown both crystallographically and in solution [3-7]. We report the crystal structure of a complex formed between IgE-Fc and two bound Fab fragments of a inhibitory anti-IgE antibody, and show that IgE-Fc can also adopt a totally extended conformation. The IgE-Fc has adopted a completely symmetrical conformation that requires an “unbending” of approximately 120° from the previously characterised structure. Molecular dynamics simulation reveals a series of stable conformations for free IgE-Fc that suggest a pathway from the acutely bent crystal structure through stable, extended conformations close to that seen in the Fab complex. We show by ITC, stoppedflow kinetic and FRET analyses that IgE-Fc adopts extended conformations in solution, and that these are an intrinsic property of IgEFc, not induced by Fab binding. We propose that IgE-Fc passes through these extended conformations as it flips between two bent conformations in which the CΣ2 domains fold back on opposite faces of the CΣ3-CΣ4 domains. The ability of IgE to exist in both bent and extended conformations may be essential for allergen recognition by IgE-Fc when bound to FcΣRI on the surface of mast cells, and as the B cell receptor respectively. Understanding the full range of conformations accessible to the free IgE molecule is also key to developing IgE-targeted therapies for allergic disease.