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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 intensity ratios and chemical shift changes of 15N-labelled RXFP1(1–72) titrated with H2 relaxin or analogues.(a,b) H2 relaxin, (c,d) A-chain (9–24) H2 relaxin and (e,f) H2/I5-2 H2 chimera. In (a,c,e) (red circles) represent 50 μM 15N-labelled RXFP1(1–72) in the presence of 0.2 μM Mn2+-DTPA-(A)-H2; (black squares) indicate changes to the intensity ratio following addition of one molar equivalent H2 relaxin or analogue. (b,d,f) show the change in average 1HN and 15N chemical shifts after titration of the RXFP1(1–72) and Mn2+-DTPA-(A)-H2 complex with one molar equivalent of H2 relaxin or analogue. 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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f4: Plots of intensity ratios and chemical shift changes of 15N-labelled RXFP1(1–72) titrated with H2 relaxin or analogues.(a,b) H2 relaxin, (c,d) A-chain (9–24) H2 relaxin and (e,f) H2/I5-2 H2 chimera. In (a,c,e) (red circles) represent 50 μM 15N-labelled RXFP1(1–72) in the presence of 0.2 μM Mn2+-DTPA-(A)-H2; (black squares) indicate changes to the intensity ratio following addition of one molar equivalent H2 relaxin or analogue. (b,d,f) show the change in average 1HN and 15N chemical shifts after titration of the RXFP1(1–72) and Mn2+-DTPA-(A)-H2 complex with one molar equivalent of H2 relaxin or analogue. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.

Mentions: H2 relaxin has a propensity to dimerize at high concentrations18, therefore we probed the interaction of RXFP1(1–72) with substoichiometric concentrations of paramagnetically labelled H2 relaxin. As previously demonstrated19, the use of a diethylene triamine pentaacetic acid (DTPA) cage attached to the N-terminus of the A-chain of H2 relaxin does not perturb binding to RXFP1 when loaded with Eu3+. Replacement of Eu3+ with Mn2+ (Mn2+-DTPA-(A)-H2) showed no difference on binding to or activating RXFP1. Therefore we monitored paramagnetic (Mn2+) induced line-broadening in 1H-15N HSQC by titrating 50 μM 15N-labelled RXFP1(1–72) with Mn2+-DTPA-(A)-H2 (0–0.3 μM). The most significant effects were localized to Asp36 to Trp46 which comprise the C-terminus of the LDLa module and the first six residues of the linker (Fig. 4a). Considering the paramagnetic radius of Mn2+, broadening of nuclei can be up to 34 Å from the paramagnetic probe20. The observed broadening is localized and specific, suggesting that this region is in close proximity to the DTPA cage attached at the N-terminus of the A-chain. Importantly, the resonances from Asp51 to Thr61, which show the largest chemical shift changes on H2 relaxin titration, were not broadened (Figs 3a and 4a) indicating that these residues are distant from the cage and the A-chain N-terminus.


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 intensity ratios and chemical shift changes of 15N-labelled RXFP1(1–72) titrated with H2 relaxin or analogues.(a,b) H2 relaxin, (c,d) A-chain (9–24) H2 relaxin and (e,f) H2/I5-2 H2 chimera. In (a,c,e) (red circles) represent 50 μM 15N-labelled RXFP1(1–72) in the presence of 0.2 μM Mn2+-DTPA-(A)-H2; (black squares) indicate changes to the intensity ratio following addition of one molar equivalent H2 relaxin or analogue. (b,d,f) show the change in average 1HN and 15N chemical shifts after titration of the RXFP1(1–72) and Mn2+-DTPA-(A)-H2 complex with one molar equivalent of H2 relaxin or analogue. 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

f4: Plots of intensity ratios and chemical shift changes of 15N-labelled RXFP1(1–72) titrated with H2 relaxin or analogues.(a,b) H2 relaxin, (c,d) A-chain (9–24) H2 relaxin and (e,f) H2/I5-2 H2 chimera. In (a,c,e) (red circles) represent 50 μM 15N-labelled RXFP1(1–72) in the presence of 0.2 μM Mn2+-DTPA-(A)-H2; (black squares) indicate changes to the intensity ratio following addition of one molar equivalent H2 relaxin or analogue. (b,d,f) show the change in average 1HN and 15N chemical shifts after titration of the RXFP1(1–72) and Mn2+-DTPA-(A)-H2 complex with one molar equivalent of H2 relaxin or analogue. Experiments were conducted at pH 6.8 and 25 °C. Error bars represent the average estimated experimental noise for the respective NMR experiment.
Mentions: H2 relaxin has a propensity to dimerize at high concentrations18, therefore we probed the interaction of RXFP1(1–72) with substoichiometric concentrations of paramagnetically labelled H2 relaxin. As previously demonstrated19, the use of a diethylene triamine pentaacetic acid (DTPA) cage attached to the N-terminus of the A-chain of H2 relaxin does not perturb binding to RXFP1 when loaded with Eu3+. Replacement of Eu3+ with Mn2+ (Mn2+-DTPA-(A)-H2) showed no difference on binding to or activating RXFP1. Therefore we monitored paramagnetic (Mn2+) induced line-broadening in 1H-15N HSQC by titrating 50 μM 15N-labelled RXFP1(1–72) with Mn2+-DTPA-(A)-H2 (0–0.3 μM). The most significant effects were localized to Asp36 to Trp46 which comprise the C-terminus of the LDLa module and the first six residues of the linker (Fig. 4a). Considering the paramagnetic radius of Mn2+, broadening of nuclei can be up to 34 Å from the paramagnetic probe20. The observed broadening is localized and specific, suggesting that this region is in close proximity to the DTPA cage attached at the N-terminus of the A-chain. Importantly, the resonances from Asp51 to Thr61, which show the largest chemical shift changes on H2 relaxin titration, were not broadened (Figs 3a and 4a) indicating that these residues are distant from the cage and the A-chain N-terminus.

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