Structural Insights into the Action of Relaxin Peptide ...

Proceedings of the 4th International Peptide Symposium in conjunction with the 7th Australian Peptide Conference and the 2nd Asia-Pacific International Peptide Symposium, 2007

Jackie Wilce (Editor) on behalf of the Australian Peptide Association

Structural Insights into the Action of Relaxin Peptide Hormones K. Johan Rosengren1*, Ross A.D. Bathgate2, David J. Craik3, Norelle L. Daly3, Linda M. Haugaard-Jönsson1, M. Akther Hossain2 , Feng Lin2, and John D. Wade2 1

School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, SE-391 82, Sweden; 2Howard Florey Institute, University of Melbourne, Melbourne, VIC 3010, Australia; 3Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. E-mail: [email protected]

Introduction

The human relaxin family of peptide hormones comprises seven members: H1, H2 and H3 relaxin and the insulin-like peptides INSL3-6. These hormones play a number of distinct physiological roles, many of which are yet to be characterized, but they all have the common structural characteristics of two peptide chains that are folded tightly together into a compact globular fold that is stabilized by three disulfide bonds (Fig. 1).

G-protein coupled receptors (GPCRs) [4,5,6]. The H2 relaxin and INSL3 receptors LGR7 and LGR8 belong to the Leucine-rich repeat containing GPCRs, which have a large N-terminal ligand binding domain, while the H3 relaxin and INSL5 receptors belong to the classic peptide ligand GPCR family. Despite these differences, interesting cross-reactivity is observed between hormones and receptors, with H3 relaxin being able to active three of the four currently identified receptors (Fig. 2).

Fig. 2. Relaxin hormone-receptor pairs. Block arrows represent the interaction between the relaxins and their endogenous receptors, with additional cross-reactivity indicated by thin arrows. Fig. 1. Sequence alignment of the seven members of the human relaxin family. The conserved cysteines are shown in yellow and their connectivities are indicated by black lines.

H2 relaxin is the human ortholog of the mammalian relaxin hormone that was first isolated over 80 years ago and which has long been regarded as a hormone associated with pregnancy [1]. Its actions include the remodeling of the connective tissue of the reproductive tract during pregnancy, but it does have a number of other physiological roles not related to pregnancy, including regulation of collagen and vasodilatation of various tissues. INSL3 similarly plays a role in reproduction, the highest production being found in the testis and ovaries, and a key role is the initiation of testes decent during fetal development. In contrast, the most recently discovered member of the relaxin family, H3 relaxin [2], is primarily expressed in the brain and has been suggested to play a role in the neurological signaling of stress responses in the central nervous system [3]. For the remaining members of the family the physiological roles are largely unknown. Relaxins, unlike their structural relatives insulin and the insulin-like growth factors, which interact with tyrosine kinase receptors, activate two unrelated classes of

These observations raise the questions: What features are needed for interacting with the various receptors and what is the nature of these interactions? Here we have employed nuclear magnetic resonance (NMR) spectroscopy to characterize the structural differences of the relaxin hormones in order to gain new insights into how differences in the primary sequence can alter the three-dimensional structure and, as a result, the ability of the peptides to interact with the various receptors.

Results and Discussion

The overall aim of this work is to develop a structural understanding of how relaxins differ in structure and how these differences affect the ability to interact with their various receptors. Such an understanding will facilitate the design of relaxin analogues that are selective agonists or antagonist for each of the individual receptors. These analogues may be invaluable for pharmaceutical applications, as relaxins have great potential as drugs and as pharmacological probes to help deduce the in vivo functions of the various relaxins. Peptide Synthesis – The A- and B-chains of the various relaxins used for structural studies were produced by FMOC solid-phase peptide synthesis. In most cases a 1



strategy for directed formation of the three disulfide bonds was used to achieve high yields of the correctly folded products. This method involves protection of pairs of cysteine residues that can be selectively removed in a step-wise fashion, with each step followed by oxidation of the produced free thiols [7]. The peptides were purified and characterized by RP-HPLC and mass spectrometry. Structural Studies – H2 relaxin, H3 relaxin and INSL3 have all been subjected to extensive analysis by two-dimensional homonuclear 1H-NMR. The spectral data for all three peptides are of high quality with an excellent signal dispersion confirming a well-ordered structure (Fig. 3). However, a number of residues show severe broadening, suggesting that there are internal dynamic processes within the relaxin fold. Interestingly this broadening seems to be located in the same region of all studied relaxins, centered on the CysA10-CysA15 intra-chain disulfide bond. This suggests that it may be caused by a reorientation of the disulfide bond that results in fluctuating chemical shifts for the surrounding resonances. Despite this broadening, near complete assignments could be achieved for all three peptides using standard sequential assignment strategies. Fig 4. Secondary Hα shifts of relaxins A (a) and B (b) chains. Helical regions are indicated by schematic helices.

Fig. 3. NOESY spectrum recorded of H2 relaxin at 298K, pH 4 and 600 MHz with a mixing time of 150 ms. The sequential walk for the C-terminal half of the A-chain is indicated with connecting lines and residue numbers.

Secondary Hα shifts are highly sensitive to the local conformation of the peptide chain and thus give a good indication of the secondary structure present in a protein. Fig. 4 shows a comparison of the Hα secondary shifts of the three peptides and confirms that all have a similar typical relaxin fold with three helical segments, indicated by a series of negative shifts, and two short extended stretches, seen as sequences of positive shifts, forming a small anti-parallel β-sheet. From the NMR data of H3 relaxin and INSL3 we collected a large number of structural restraints, which

enabled calculation of the three-dimensional structure in solution. These restraints included inter-proton distances derived from NOE intensities in NOESY spectra, dihedral angle restraints based on coupling constants and hydrogen bond restraints for amides that were found to be involved in hydrogen bonds based on either slow exchange rates with the solvent or small effects of temperature on the amide chemical shift (temperature coefficients). The NMR data were combined with information about the covalent structure and geometry of the peptide chain and used to calculate the structure of INSL3 and H3 relaxin in solution using simulated annealing. The preliminary structures generated from these calculations were subsequently refined and energy minimized in a water-shell within the program CNS. For each peptide 50 structures were calculated and from these a family of 20 lowest energy structures that were in good agreement with the data and good covalent geometry were selected to represent the solution structures of H3 relaxin [8] and INSL3 [9]. The structures are presented in Fig. 5. When comparing the relaxin structures (Fig. 5 and Fig. 6) it is obvious that they all share a common fold with the main differences located in the termini regions. All

Fig. 4. Structures of H3 relaxin (left) and INSL3 (right). 2



Fig. 6. Relaxin structures. Comparison of the 3D structures of H3 relaxin (a), INSL3 (b), and H2 relaxin (c).

structures comprise three helical segments. The A-chain is roughly U shaped with its two helical segments running parallel to each other. The B-chain helix lies across the face of the U, perpendicular to the two A-chain helices. The N-terminal of the A-chain is structured to various degrees in relaxins with H2 and H3 relaxin having a fully helical structure, while INSL3 is unstructured up to residue five. The N-terminal helix of the A-chain is the most dynamic of the three helices, lacking slow exchanging amides and comprising the CysA10-CysA15 disulfide bond, which is sampling different conformations. The B-chain N-terminal appears to be fairly disordered in all structures. By contrast, the crystal structure of H2 displays a short 310 helix in this region but this is likely to be a result of crystal packing. No long range NOEs are seen in this region suggesting any interactions with the rest of the molecule. The B-chain helical region represents the main receptor biding interface with the Arg-x-x-x-Arg-x-x-Val motif of H2 and H3 relaxin being crucial for activity. We recently showed that a similar motif comprising HisB12, ArgB16, ValB19 and ArgB20 is involved in the receptor binding of INSL3 [9]. In addition it has previously been established that TrpB27 is crucial for the action of INSL3 and it was recently shown that this is also the case for H3 relaxin. Thus the conformation of the B-chain C-termini of the hormones is particularly interesting. In the H2 relaxin crystal structure [10] the Trp extends away from the molecular core but in contrast our studies have shown that in H3 relaxin and INSL3 the Trp may fold back to form interactions with the molecular core. These interactions are confirmed by a number of NOE contacts involving the Trp aromatic side chain. Although this would suggest that its preferred orientation may be packed up against the core the fact that the NOEs are weak in strength and the small deviations from random coil seen in this area suggest the tail is still flexible and is likely to adopt a different conformation when binding to its receptor.

Conclusions

The relaxin family of peptide hormones all share a common fold with the main difference being around the termini regions. The fold is considerably flexible as evident from line broadening of a number of residues and

the poor definition of the structure in certain areas. This is interesting as flexibility is in many cases important for function as the peptide may need to adjust its conformation upon binding. The B-chain C-termini, which is crucial for receptor interaction for both H3 relaxin and INSL3, appears from chemical shift analysis to be fairly unstructured but for all peptides some NOEs can be detected between the C-terminal Trp and the peptide core. It is possible that differences in the degree of flexibility and preferred orientation may be contributing to the differences in receptor selectivity.

Acknowledgments This work was supported by the Univeristy of Kalmar (KJR), Åke Wibergs Foundation (KJR), and NHMRC Australia (RAB and JDW) NLD is an NHMRC Industry Fellow. DJC is an ARC Professorial Fellow.

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