Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels.

Jeffrey R Simmons,Alexandre Murza,Éric Marsault,Calem Kenward,Michael D Lumsden,Jan K Rainey

doi:10.3390/ijms20153658

Jeffrey R Simmons, Alexandre Murza + Show 4 more

Open Access

https://doi.org/10.3390/ijms20153658

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Abstract

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on 13C- and/or 15N-enrichment alongside 1H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of 19F nuclei into a sample. The 19F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by 19F NMR. Distinct ranges of 19F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive 19F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species.

Highlights

The apelinergic system has been implicated in wide-ranging physiological processes, including vasodilation, cardiovascular development, and insulin homeostasis [1,2]
The 19F chemical shift ranges observed for the 2,4,5F-Phe in buffer, micelles, and in titration with
It should be noted that F-Trp chemical shifts may vary

Summary

Introduction

The apelinergic system has been implicated in wide-ranging physiological processes, including vasodilation, cardiovascular development, and insulin homeostasis [1,2]. E19Fadcdhiteimoniacla,lshshieilfdtepderretsuornbaantcioe.nb(RΔeδp)orretleadtiavsethtoe control range of sample in absence perturbation relative of to binding partner the original peak mfoarxiAmRum55.c:F∆*δ-adpe-t1er3mininteedraucstiinogncse.nter of the two peaks that become apparent upon ligand addition (Figure 6a; red spectrum in the left panel) relative to unliganded form (Figure 6a; blue spectrum in the left panel) since unambiguous assignment of each to a given Trp is not possible. E to the original peak maximum.c Δδ determined using center of the two peaks that become apparent upon ligand addition (Figure 6a; red spectrum in the left panel) relative to unliganded form (Figure 6a; blue spectrum in the left panel) since unambiguous assignment of each to a given Trp is not possible. Septreyct(rgaraereenc);oamnpda,r2e:d1 floigraenadch:rescpeepciteosrasltooniceh(ibolmuee)t;rayt (1p:2ulripglaen)d. : receptor stoichiometry (red); 1:1 stoichiometry (green); and, 2:1 ligand:receptor stoichiometry (purple)

Discussion

Findings

Apelin Receptor Fragments