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Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

ABSTRACT: The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg²³ Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg¹², Ile¹⁵, and Ile¹⁹ Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe¹⁴ and Ala¹⁷, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

SUBMITTER: Haugaard-Kedstrom LM

PROVIDER: S-EPMC6187623 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

Haugaard-Kedström Linda M LM Lee Han Siean HS Jones Maryon V MV Song Angela A Rathod Vishaal V Hossain Mohammed Akhter MA Bathgate Ross A D RAD Rosengren K Johan KJ

The Journal of biological chemistry 20180821 41

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically charac ...[more]

PMID: 30131342

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Project description:G-protein coupled receptors (GPCR) transduce extracellular stimuli into the cell interior and are thus centrally involved in almost all physiological-neuronal processes. This essential function and association with many diseases or pathological conditions explain why GPCRs are one of the priority targets in medical and pharmacological research, including structure determination. Despite enormous experimental efforts over the last decade, both the expression and purification of these membrane proteins remain elusive. This is attributable to specificities of each GPCR subtype and the finding of necessary experimental in vitro conditions, such as expression in heterologous cell systems or with accessory proteins. One of these specific GPCRs is the leucine-rich repeat domain (LRRD) containing GPCR 7 (LGR7), also termed relaxin family peptide receptor 1 (RXFP1). This receptor is characterized by a large extracellular region of around 400 amino acids constituted by several domains, a rare feature among rhodopsin-like (class A) GPCRs. In the present study, we describe the expression and purification of RXFP1, including the design of various constructs suitable for functional/biophysical studies and structure determination. Based on available sequence information, homology models, and modern biochemical and genetic tools, several receptor variations with different purification tags and fusion proteins were prepared and expressed in Sf9 cells (small-scale), followed by an analytic fluorescence-detection size-exclusion chromatography (F-SEC) to evaluate the constructs. The most promising candidates were expressed and purified on a large-scale, accompanied by ligand binding studies using surface plasmon resonance spectroscopy (SPR) and by determination of signaling capacities. The results may support extended studies on RXFP1 receptor constructs serving as targets for small molecule ligand screening or structural elucidation by protein X-ray crystallography or cryo-electron microscopy.

Dataset Information

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

Publications

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

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