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Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

ABSTRACT: The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.

SUBMITTER: Wingler LM

PROVIDER: S-EPMC6367718 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

Wingler Laura M LM McMahon Conor C Staus Dean P DP Lefkowitz Robert J RJ Kruse Andrew C AC

Cell 20190110 3

The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular ...[more]

PMID: 30639100

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Project description:Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin-1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (Gs, Gq and β-arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A-mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb-NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with Gs signaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand-induced upstream Gq signaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.

Dataset Information

Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

Publications

Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

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