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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.


Chemical shift differences following a titration of 15N-labelled RXFP1(1–72) with 20 molar equivalents of EL1(475–486)/EL2-RXFP1.(a) portion of 1H-15N HSQC spectrum of RXFP1(1–72) showing chemical shift effects on titration with EL1(475–486)/EL2-RXFP1 (b) chemical shift differences for wild-type RXFP(1–72) (black), F54A (blue) and G41A/D42A (red) (c) wild-type RXFP(1–72) (black), G45A/W46A (blue) and C40Ains (red). Experiments were conducted at pH 6.8 and 25 °C.
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f7: Chemical shift differences following a titration of 15N-labelled RXFP1(1–72) with 20 molar equivalents of EL1(475–486)/EL2-RXFP1.(a) portion of 1H-15N HSQC spectrum of RXFP1(1–72) showing chemical shift effects on titration with EL1(475–486)/EL2-RXFP1 (b) chemical shift differences for wild-type RXFP(1–72) (black), F54A (blue) and G41A/D42A (red) (c) wild-type RXFP(1–72) (black), G45A/W46A (blue) and C40Ains (red). Experiments were conducted at pH 6.8 and 25 °C.

Mentions: Measuring the molecular interactions of H2 relaxin or LDLa-linker with full-length RXFP1 or its TMD is confounded by the multistep mode of activation2829. While the LDLa module is indispensible for receptor activation11, the mutants of the LDLa-LRR linker presented here suggest critical roles of Asp42, Asn43, Gly45 and Trp46 in H2-mediated receptor activation (Table 1), with no direct involvement in H2 relaxin binding (Figs 2 and 6). This led us to investigate if the LDLa-LRR linker interacts with the exoloops of the TMD of RXFP1. To probe H2 relaxin and LDLa interactions with the exoloops of the TMD we have engineered a soluble protein scaffold where we graft exoloop-1 and -2 of the TMD onto the backbone of a thermostabilized version of the B1 immunoglobulin binding domain of streptococcal protein G (GB1) (ref. 14). Here we use a similar scaffold comprised of the entire exoloop-2 (Glu551 to Gln575) and the region of exoloop-1 (Ala475 to Gln486). This construct (EL1(475–486)/EL2-GB1) is similar to our previously reported EL1/EL2-GB1, except EL1 has been truncated which improves its expression and solubility. EL1(475–486)/EL2-GB1 maintains the disulfide between the C-terminal end of exoloop-1 and the centre of exoloop-2 that is essential for structure and function30. Titration of 15N-labelled RXFP1(1–72) with EL1(475–486)/EL2-GB1 showed chemical shift changes for Gly41, Ser47 to Asp51, Ala55 and Lys59 to Thr61 (Fig. 7a,b) suggesting that the LDLa-LRR linker may interact with the ELs of the TMD. To test the specificity of side-chain interactions, the key linker mutations C40Ains, G41A/D42A, G45A/W46A and F50A RXFP1(1–72) were selected for titration against EL1(475–486)/EL2-RXFP1. None of these mutants showed an interaction with EL1(475–486)/EL2-RXFP1 (Fig. 7b,c). However, the mutant, F54A-RXFP1(1–72), which showed significant loss of H2 relaxin binding, but modest loss of activation, could still interact with EL1(475–486)/EL2-GB1 (Fig. 7b).


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)

Chemical shift differences following a titration of 15N-labelled RXFP1(1–72) with 20 molar equivalents of EL1(475–486)/EL2-RXFP1.(a) portion of 1H-15N HSQC spectrum of RXFP1(1–72) showing chemical shift effects on titration with EL1(475–486)/EL2-RXFP1 (b) chemical shift differences for wild-type RXFP(1–72) (black), F54A (blue) and G41A/D42A (red) (c) wild-type RXFP(1–72) (black), G45A/W46A (blue) and C40Ains (red). Experiments were conducted at pH 6.8 and 25 °C.
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f7: Chemical shift differences following a titration of 15N-labelled RXFP1(1–72) with 20 molar equivalents of EL1(475–486)/EL2-RXFP1.(a) portion of 1H-15N HSQC spectrum of RXFP1(1–72) showing chemical shift effects on titration with EL1(475–486)/EL2-RXFP1 (b) chemical shift differences for wild-type RXFP(1–72) (black), F54A (blue) and G41A/D42A (red) (c) wild-type RXFP(1–72) (black), G45A/W46A (blue) and C40Ains (red). Experiments were conducted at pH 6.8 and 25 °C.
Mentions: Measuring the molecular interactions of H2 relaxin or LDLa-linker with full-length RXFP1 or its TMD is confounded by the multistep mode of activation2829. While the LDLa module is indispensible for receptor activation11, the mutants of the LDLa-LRR linker presented here suggest critical roles of Asp42, Asn43, Gly45 and Trp46 in H2-mediated receptor activation (Table 1), with no direct involvement in H2 relaxin binding (Figs 2 and 6). This led us to investigate if the LDLa-LRR linker interacts with the exoloops of the TMD of RXFP1. To probe H2 relaxin and LDLa interactions with the exoloops of the TMD we have engineered a soluble protein scaffold where we graft exoloop-1 and -2 of the TMD onto the backbone of a thermostabilized version of the B1 immunoglobulin binding domain of streptococcal protein G (GB1) (ref. 14). Here we use a similar scaffold comprised of the entire exoloop-2 (Glu551 to Gln575) and the region of exoloop-1 (Ala475 to Gln486). This construct (EL1(475–486)/EL2-GB1) is similar to our previously reported EL1/EL2-GB1, except EL1 has been truncated which improves its expression and solubility. EL1(475–486)/EL2-GB1 maintains the disulfide between the C-terminal end of exoloop-1 and the centre of exoloop-2 that is essential for structure and function30. Titration of 15N-labelled RXFP1(1–72) with EL1(475–486)/EL2-GB1 showed chemical shift changes for Gly41, Ser47 to Asp51, Ala55 and Lys59 to Thr61 (Fig. 7a,b) suggesting that the LDLa-LRR linker may interact with the ELs of the TMD. To test the specificity of side-chain interactions, the key linker mutations C40Ains, G41A/D42A, G45A/W46A and F50A RXFP1(1–72) were selected for titration against EL1(475–486)/EL2-RXFP1. None of these mutants showed an interaction with EL1(475–486)/EL2-RXFP1 (Fig. 7b,c). However, the mutant, F54A-RXFP1(1–72), which showed significant loss of H2 relaxin binding, but modest loss of activation, could still interact with EL1(475–486)/EL2-GB1 (Fig. 7b).

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.