The Relaxin Receptor-binding Site Geometry Suggests a Novel Gripping Mode of Interaction

Abstract

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain ␣-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40°. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with ␤-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby ␤-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.Relaxin, a small, two-chain protein, the physiological mediator of parturition in most mammals (1), has recently been shown to influence significantly the symptoms of scleroderma (2, 3). Since its discovery (4) relaxin has provided a share of unusual features including an insulin-like structure (5-9) and a receptor-binding site composed of two charged residues, i.e. arginine in position B13 and B17 (10, 11). The binding residues are positioned one turn apart on the major B chain helix and are projecting parallel into the surrounding water (12). This observation led to the suggestion that relaxin would bind to the receptor by a dual prong mechanism involving the interaction of the guanidinium groups with two negative charges at the bottom of a binding pocket in the receptor (11). Although both arginines are indispensable, the fact that arginine-containing peptides would not interfere with binding suggested that other binding site members might exist. In this paper, we are reporting that the receptor-binding site of relaxin includes isoleucine in position B20, which is located three-quarter of one turn further toward the C-terminal end of the same helix so that the hydrophobic side chain opposes the two arginines forming a quasi-prehensile unit that points to a novel binding mechanism. Evidence presented in this paper supports the conclusion that Ile-B20 is as important for receptor-binding as either of the critical arginines and that the relaxin/receptor interaction is trivalent. EXPERIMENTAL PROCEDURES MaterialsA...

R3(BΔ23–27)R/I5 Chimeric Peptide, a Selective Antagonist for GPCR135 and GPCR142 over Relaxin Receptor LGR7

“…However, an Ile 19 to Glu (the corresponding residue at the B-chain of INSL6 (40)) change not only abolished the activity of relaxin-3 for LGR7 but also decreased relaxin-3 activity for GPCR135 and GPCR142 dramatically, suggesting that INSL6 might not be a ligand for GPCR135, GPCR142, or LGR7. These results further support the proposition that R 12 EFIR 16 AVI 19 is serving as the RXXXRXXI motif for LGR7 binding (34).…”

Structure of the R3/I5 Chimeric Relaxin Peptide, a Selective GPCR135 and GPCR142 Agonist

“…The answer is that the native B-chain structure is fully retained, which allows for several conclusions to be drawn regarding the receptor interactions. It has been established that the primary binding site of relaxin for LGR7 is located within the R-X-X-X-R-X-X-I motif that is presented on one face of the B-chain helix (24), and recent mutational explorations of the relaxin-3 B-chain have shown that the same motif is crucial for the binding of relaxin-3 to both GPCR135 and LGR7 (21). The observation that the B-chain structure, including the receptor binding motif, is retained in the chimera thus provides a clear explanation for the high affinity binding to the GPCR135 and GPCR142 receptors, and is consistent with the idea of the B-chain alone carrying all structural features needed for activation of these receptors.…”

“…From these structures together with the crystal structure of relaxin-2 and extensive structure-activity studies, a picture of the complex events involved in the recognition of a relaxin by its receptor is starting to emerge. A conserved binding motif Arg-X-X-X-Arg-X-X-Ile has been identified as a crucial interaction point for relaxin binding to LGR7 (24), and a similar motif (His-X-X-X-Arg-X-X-Val) is used by INSL3 for binding to its receptor (23). However, it is clear that several additional interactions are needed both for strong binding and subsequent activation of the receptors.…”

Characterization and biological activity of relaxin in porcine milk

“…Further processing of the connecting peptide results in the native 6 kDa RLX molecule found in circulation (O'Byrne et al 1989). The amino acid motif Arg-X-X-X-Arg-X-X-Ile, located in the middle of the B chain, is required for RLX bioactivity (Bullesbach & Schwabe 2000).…”