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The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

Sethi A, Bruell S, Patil N, Hossain MA, Scott DJ, Petrie EJ, Bathgate RA, Gooley PR - Nat Commun (2016)

Bottom Line: H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1.Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation.We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry &Molecular Biology, The University of Melbourne, Victoria 3010, Australia.

ABSTRACT
H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

No MeSH data available.


Related in: MedlinePlus

Titration of 50 μM 15N-labelled RXFP1(1–72) with H2 relaxin.(a) Plot of the change in average 1HN and 15N chemical shifts and (b) peak intensities following titration of 15N-RXFP1(1–72) with nine equivalents of H2 relaxin. (c) Representative region of the 1H,15N HSQC spectrum showing chemical shift dependence on H2 relaxin. (d) Single-site saturation binding curves (Kd=200±10 μM) for the three resonances that show the largest chemical shift changes and remained resolved throughout the titration. Experiments were conducted at pH 6.8 and 25 °C.
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f3: Titration of 50 μM 15N-labelled RXFP1(1–72) with H2 relaxin.(a) Plot of the change in average 1HN and 15N chemical shifts and (b) peak intensities following titration of 15N-RXFP1(1–72) with nine equivalents of H2 relaxin. (c) Representative region of the 1H,15N HSQC spectrum showing chemical shift dependence on H2 relaxin. (d) Single-site saturation binding curves (Kd=200±10 μM) for the three resonances that show the largest chemical shift changes and remained resolved throughout the titration. Experiments were conducted at pH 6.8 and 25 °C.

Mentions: The weakening of H2 relaxin binding by mutations within the region GDNNGW was unexpected. To gain detailed insight into the role of the linker in H2 relaxin binding, we recombinantly expressed and purified fractionally deuterated 13C,15N-labelled LDLa module with the 32 residues of the linker, designated RXFP1(1–72) and assigned all the backbone resonances (Supplementary Fig. 1). To investigate the interaction between RXFP1(1–72) and H2 relaxin, we performed a two-dimensional (2D) 1H-15N HSQC monitored H2 relaxin titration of 15N-labelled RXFP1(1–72). Significant chemical shift and intensity differences were noted for residues assigned to the linker region comprising Trp46 to Gln63 (Fig. 3a,b). From the chemical shift difference plot, Asp51, Ala55, Tyr57 and Thr61 (Fig. 3a,c) experienced the largest chemical shift changes. Fitting these differences of Asp51, Ala55 and Thr61, which remain resolved in the 1H-15N HSQC spectra throughout the titration, to a single-site binding curve shows the affinity of H2 relaxin for RXFP1(1–72) is 200±10 μM (Fig. 3d). Importantly, residues within the LDLa module are largely unperturbed and residues from Gly41 to Asn45 also show minimal chemical shift and intensity changes, suggesting that although mutation to this region perturbs activity it does not directly bind H2 relaxin.


The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

Sethi A, Bruell S, Patil N, Hossain MA, Scott DJ, Petrie EJ, Bathgate RA, Gooley PR - Nat Commun (2016)

Titration of 50 μM 15N-labelled RXFP1(1–72) with H2 relaxin.(a) Plot of the change in average 1HN and 15N chemical shifts and (b) peak intensities following titration of 15N-RXFP1(1–72) with nine equivalents of H2 relaxin. (c) Representative region of the 1H,15N HSQC spectrum showing chemical shift dependence on H2 relaxin. (d) Single-site saturation binding curves (Kd=200±10 μM) for the three resonances that show the largest chemical shift changes and remained resolved throughout the titration. Experiments were conducted at pH 6.8 and 25 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837482&req=5

f3: Titration of 50 μM 15N-labelled RXFP1(1–72) with H2 relaxin.(a) Plot of the change in average 1HN and 15N chemical shifts and (b) peak intensities following titration of 15N-RXFP1(1–72) with nine equivalents of H2 relaxin. (c) Representative region of the 1H,15N HSQC spectrum showing chemical shift dependence on H2 relaxin. (d) Single-site saturation binding curves (Kd=200±10 μM) for the three resonances that show the largest chemical shift changes and remained resolved throughout the titration. Experiments were conducted at pH 6.8 and 25 °C.
Mentions: The weakening of H2 relaxin binding by mutations within the region GDNNGW was unexpected. To gain detailed insight into the role of the linker in H2 relaxin binding, we recombinantly expressed and purified fractionally deuterated 13C,15N-labelled LDLa module with the 32 residues of the linker, designated RXFP1(1–72) and assigned all the backbone resonances (Supplementary Fig. 1). To investigate the interaction between RXFP1(1–72) and H2 relaxin, we performed a two-dimensional (2D) 1H-15N HSQC monitored H2 relaxin titration of 15N-labelled RXFP1(1–72). Significant chemical shift and intensity differences were noted for residues assigned to the linker region comprising Trp46 to Gln63 (Fig. 3a,b). From the chemical shift difference plot, Asp51, Ala55, Tyr57 and Thr61 (Fig. 3a,c) experienced the largest chemical shift changes. Fitting these differences of Asp51, Ala55 and Thr61, which remain resolved in the 1H-15N HSQC spectra throughout the titration, to a single-site binding curve shows the affinity of H2 relaxin for RXFP1(1–72) is 200±10 μM (Fig. 3d). Importantly, residues within the LDLa module are largely unperturbed and residues from Gly41 to Asn45 also show minimal chemical shift and intensity changes, suggesting that although mutation to this region perturbs activity it does not directly bind H2 relaxin.

Bottom Line: H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1.Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation.We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry &Molecular Biology, The University of Melbourne, Victoria 3010, Australia.

ABSTRACT
H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.

No MeSH data available.


Related in: MedlinePlus