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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

Plots of 13Cαβ secondary chemical shifts and 15N{1H}-NOEs of RXFP1(1–72) in the absence (black) and presence (red) of three molar equivalents of H2 relaxin.(a) Deviations of the measured 13Cα and 13Cβ chemical shifts from random coil where persistent positive or negative deviations indicate the presence of α-helix or β-strand, respectively. Above this plot is schematically represented the known secondary structure of the LDLa module and included a region of α-helix that increases in the presence of H2 relaxin to spanning Leu48 to Ser56. (b) Plot of 15N{1H}-NOEs that shows on addition of relaxin the 15N{1H}-NOEs increase for the region Gly41 to Lys59. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.
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f5: Plots of 13Cαβ secondary chemical shifts and 15N{1H}-NOEs of RXFP1(1–72) in the absence (black) and presence (red) of three molar equivalents of H2 relaxin.(a) Deviations of the measured 13Cα and 13Cβ chemical shifts from random coil where persistent positive or negative deviations indicate the presence of α-helix or β-strand, respectively. Above this plot is schematically represented the known secondary structure of the LDLa module and included a region of α-helix that increases in the presence of H2 relaxin to spanning Leu48 to Ser56. (b) Plot of 15N{1H}-NOEs that shows on addition of relaxin the 15N{1H}-NOEs increase for the region Gly41 to Lys59. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.

Mentions: 15N and 13C-edited NOESY spectra do not show any stable structure within the linker region for RXFP1(1–72). However, 13Cα and 13Cβ are sensitive to the presence of secondary structure in proteins, including intrinsically unstructured proteins2425. Indeed for RXFP1(1–72) we observe the persistence of positive and negative (ΔCα—ΔCβ) smoothed values26 consistent with the expected helical and β-strand structure of the LDLa module12. In the absence of H2 relaxin, the (ΔCα—ΔCβ) smoothed values suggest that the linker is largely unstructured, except for a region of positive values that point to the presence of a turn of α-helix comprising residues Gln49 to Lys52 (Fig. 5a). On titration of 13C,15N-labelled RXFP1(1–72) with H2 relaxin the helical propensity of these residues increases and extends to include Leu48 to Ser56 suggesting H2 relaxin binds and stabilizes a helical structure in the linker. To further characterize the presence of residual structure, we recorded 15N{1H}-NOE experiments27 on 15N-labelled RXFP1(1–72) (Fig. 5b). The 15N{1H}-NOE values (0.74±0.08) for the LDLa module (residues 6–40) agree with a folded structure (PDB 2jm4), with a flexible N-terminus (residues 1–5, 15N{1H}-NOEs <0.4). In the linker region, a progressive decrease in 15N{1H}-NOE to 0.2 is observed for Gly41 to Trp46, suggesting increasing flexibility. However, this decrease is followed by a rise in the 15N{1H}-NOEs to 0.49±0.10 (Leu48 to Met60) consistent with the presence of residual structure across the relaxin-binding site. Following Thr61 the 15N{1H}-NOE progressively decreases, implying a flexible C-terminal region. On addition of relaxin to 15N-labelled RXFP1(1–72), little change is observed in the 15N{1H}-NOEs for residues 6–40 of the LDLa module (0.75±0.10). However, for the linker region from Gly41 to Lys59 there is a distinct increase in 15N{1H}-NOEs, from 0.47±0.11 without H2 relaxin to 0.55±0.13 with H2 relaxin, supporting stabilization of helical structure within this region.


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)

Plots of 13Cαβ secondary chemical shifts and 15N{1H}-NOEs of RXFP1(1–72) in the absence (black) and presence (red) of three molar equivalents of H2 relaxin.(a) Deviations of the measured 13Cα and 13Cβ chemical shifts from random coil where persistent positive or negative deviations indicate the presence of α-helix or β-strand, respectively. Above this plot is schematically represented the known secondary structure of the LDLa module and included a region of α-helix that increases in the presence of H2 relaxin to spanning Leu48 to Ser56. (b) Plot of 15N{1H}-NOEs that shows on addition of relaxin the 15N{1H}-NOEs increase for the region Gly41 to Lys59. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Plots of 13Cαβ secondary chemical shifts and 15N{1H}-NOEs of RXFP1(1–72) in the absence (black) and presence (red) of three molar equivalents of H2 relaxin.(a) Deviations of the measured 13Cα and 13Cβ chemical shifts from random coil where persistent positive or negative deviations indicate the presence of α-helix or β-strand, respectively. Above this plot is schematically represented the known secondary structure of the LDLa module and included a region of α-helix that increases in the presence of H2 relaxin to spanning Leu48 to Ser56. (b) Plot of 15N{1H}-NOEs that shows on addition of relaxin the 15N{1H}-NOEs increase for the region Gly41 to Lys59. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.
Mentions: 15N and 13C-edited NOESY spectra do not show any stable structure within the linker region for RXFP1(1–72). However, 13Cα and 13Cβ are sensitive to the presence of secondary structure in proteins, including intrinsically unstructured proteins2425. Indeed for RXFP1(1–72) we observe the persistence of positive and negative (ΔCα—ΔCβ) smoothed values26 consistent with the expected helical and β-strand structure of the LDLa module12. In the absence of H2 relaxin, the (ΔCα—ΔCβ) smoothed values suggest that the linker is largely unstructured, except for a region of positive values that point to the presence of a turn of α-helix comprising residues Gln49 to Lys52 (Fig. 5a). On titration of 13C,15N-labelled RXFP1(1–72) with H2 relaxin the helical propensity of these residues increases and extends to include Leu48 to Ser56 suggesting H2 relaxin binds and stabilizes a helical structure in the linker. To further characterize the presence of residual structure, we recorded 15N{1H}-NOE experiments27 on 15N-labelled RXFP1(1–72) (Fig. 5b). The 15N{1H}-NOE values (0.74±0.08) for the LDLa module (residues 6–40) agree with a folded structure (PDB 2jm4), with a flexible N-terminus (residues 1–5, 15N{1H}-NOEs <0.4). In the linker region, a progressive decrease in 15N{1H}-NOE to 0.2 is observed for Gly41 to Trp46, suggesting increasing flexibility. However, this decrease is followed by a rise in the 15N{1H}-NOEs to 0.49±0.10 (Leu48 to Met60) consistent with the presence of residual structure across the relaxin-binding site. Following Thr61 the 15N{1H}-NOE progressively decreases, implying a flexible C-terminal region. On addition of relaxin to 15N-labelled RXFP1(1–72), little change is observed in the 15N{1H}-NOEs for residues 6–40 of the LDLa module (0.75±0.10). However, for the linker region from Gly41 to Lys59 there is a distinct increase in 15N{1H}-NOEs, from 0.47±0.11 without H2 relaxin to 0.55±0.13 with H2 relaxin, supporting stabilization of helical structure within this region.

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