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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.


Comparison of binding of Mn2+-DTPA-(A)-H2, chemical shift differences and 15N{1H}-NOEs of wild type and mutants of RXFP(1–72).The first column is the difference (mutant less wild type) in peak intensities from a titration of 50 μM mutant and wild-type LDLa-linker with 0.2 μM Mn2+-DTPA-(A)-H2. A positive difference indicates less binding of Mn2+-DTPA-(A)-H2 to the mutants. The second column is the average 1HN and 15N chemical shift differences (Δδ) of mutant to wild-type protein. The third column is the difference (wild-type less mutant) of 15N{1H}-NOE of mutant and wild-type LDLa-linker. A positive difference indicates a lower 15N{1H}-NOE in the mutant. Experiments were conducted at pH 6.8 and 25 °C on (a) G41A/D42A, (b) N43A/N44A, (c) G45A/W46A, (d) F50A, and (e) F54A/Y58A. Additional mutants and data are in Supplementary Fig. 6.
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f6: Comparison of binding of Mn2+-DTPA-(A)-H2, chemical shift differences and 15N{1H}-NOEs of wild type and mutants of RXFP(1–72).The first column is the difference (mutant less wild type) in peak intensities from a titration of 50 μM mutant and wild-type LDLa-linker with 0.2 μM Mn2+-DTPA-(A)-H2. A positive difference indicates less binding of Mn2+-DTPA-(A)-H2 to the mutants. The second column is the average 1HN and 15N chemical shift differences (Δδ) of mutant to wild-type protein. The third column is the difference (wild-type less mutant) of 15N{1H}-NOE of mutant and wild-type LDLa-linker. A positive difference indicates a lower 15N{1H}-NOE in the mutant. Experiments were conducted at pH 6.8 and 25 °C on (a) G41A/D42A, (b) N43A/N44A, (c) G45A/W46A, (d) F50A, and (e) F54A/Y58A. Additional mutants and data are in Supplementary Fig. 6.

Mentions: The site-directed mutagenesis experiments on RXFP1 suggested that the first six residues within the linker (GDNNGW) are functionally important. The results from the NMR experiments conducted on RXFP1(1–72), however, suggested that these residues are not directly involved in side-chain interactions with H2 relaxin. To further understand the role of individual linker residues in the interaction with H2 relaxin we translated the mutations that had the most profound effect on RXFP1 activity and H2 relaxin binding to the recombinant RXFP1(1–72) (C40Ains, G41A/D42A, N43A/N44A, G45A/W46A, F50A, F50AIns, F54A and F54A/Y58A). We assessed the 15N and 1HN chemical shift effects of mutation, differences in their 15N{1H}-NOE profile and H2 relaxin binding by titration with Mn2+-DTPA-(A)-H2 (Fig. 6, Supplementary Fig. 6).


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)

Comparison of binding of Mn2+-DTPA-(A)-H2, chemical shift differences and 15N{1H}-NOEs of wild type and mutants of RXFP(1–72).The first column is the difference (mutant less wild type) in peak intensities from a titration of 50 μM mutant and wild-type LDLa-linker with 0.2 μM Mn2+-DTPA-(A)-H2. A positive difference indicates less binding of Mn2+-DTPA-(A)-H2 to the mutants. The second column is the average 1HN and 15N chemical shift differences (Δδ) of mutant to wild-type protein. The third column is the difference (wild-type less mutant) of 15N{1H}-NOE of mutant and wild-type LDLa-linker. A positive difference indicates a lower 15N{1H}-NOE in the mutant. Experiments were conducted at pH 6.8 and 25 °C on (a) G41A/D42A, (b) N43A/N44A, (c) G45A/W46A, (d) F50A, and (e) F54A/Y58A. Additional mutants and data are in Supplementary Fig. 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Comparison of binding of Mn2+-DTPA-(A)-H2, chemical shift differences and 15N{1H}-NOEs of wild type and mutants of RXFP(1–72).The first column is the difference (mutant less wild type) in peak intensities from a titration of 50 μM mutant and wild-type LDLa-linker with 0.2 μM Mn2+-DTPA-(A)-H2. A positive difference indicates less binding of Mn2+-DTPA-(A)-H2 to the mutants. The second column is the average 1HN and 15N chemical shift differences (Δδ) of mutant to wild-type protein. The third column is the difference (wild-type less mutant) of 15N{1H}-NOE of mutant and wild-type LDLa-linker. A positive difference indicates a lower 15N{1H}-NOE in the mutant. Experiments were conducted at pH 6.8 and 25 °C on (a) G41A/D42A, (b) N43A/N44A, (c) G45A/W46A, (d) F50A, and (e) F54A/Y58A. Additional mutants and data are in Supplementary Fig. 6.
Mentions: The site-directed mutagenesis experiments on RXFP1 suggested that the first six residues within the linker (GDNNGW) are functionally important. The results from the NMR experiments conducted on RXFP1(1–72), however, suggested that these residues are not directly involved in side-chain interactions with H2 relaxin. To further understand the role of individual linker residues in the interaction with H2 relaxin we translated the mutations that had the most profound effect on RXFP1 activity and H2 relaxin binding to the recombinant RXFP1(1–72) (C40Ains, G41A/D42A, N43A/N44A, G45A/W46A, F50A, F50AIns, F54A and F54A/Y58A). We assessed the 15N and 1HN chemical shift effects of mutation, differences in their 15N{1H}-NOE profile and H2 relaxin binding by titration with Mn2+-DTPA-(A)-H2 (Fig. 6, Supplementary Fig. 6).

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.