Solution Structure, Aggregation Behavior, and Flexibility of Human

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Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin‑2 Linda M. Haugaard-Kedström,†,□ Mohammed Akhter Hossain,§,∥ Norelle L. Daly,‡,# Ross A. D. Bathgate,§,⊥ Ernst Rinderknecht,∇ John D. Wade,§,∥ David J. Craik,‡ and K. Johan Rosengren*,†,□ †

School of Biomedical Sciences and ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia □ School of Natural Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden § Florey Institute of Neuroscience and Mental Health, ∥School of Chemistry, and ⊥Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia # Centre for Biodiscovery and Molecular Development of Therapeutics, AITHM, James Cook University, Cairns, Queensland 4878, Australia ∇ Corthera, c/o Novartis Corporation, San Carlos, California 94070, United States ABSTRACT: Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a twochain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.

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bonds, one within the A-chain and two between the chains (Figure 1A). This structural arrangement is shared with insulin and other peptides of the relaxin/insulin superfamily. In 1991, Eigenbrot et al. reported the crystal structure of H2 relaxin and showed that the peptide crystallizes as a dimer (Figure 1B).13 The dimerization behavior was confirmed by sedimentation equilibrium analytical ultracentrifugation studies, which revealed an association constant of ∼6 × 105 M−1. However, the physiological role of this self-association, if any, is unclear as porcine relaxin does not self-associate under similar conditions.14 More recently, the solution structures of H3 relaxin,15 insulin-like peptide 3 (INSL3),16 and INSL517 have been resolved using NMR spectroscopy techniques and the relaxins share a conserved fold but have local structural differences, in particular around the chain termini. None of these peptides showed significant self-association under NMR conditions.

n 1926, Frederick Hisaw identified relaxin as a pubic ligament relaxing substance in guinea pigs, and ever since, it has been considered a hormone associated with pregnancy.1 Recently, it has been appreciated that relaxin is a pleiotropic hormone involved in a number of different physiological and pathological processes including collagen regulation, allergic responses, wound healing, cardiovascular regulation, and cancer progression.2−7 Consequently, relaxin has attracted considerable interest as a pharmacologic agent.8,9 Perhaps most promising is the treatment of acute heart failure, where the human form of relaxin, H2 relaxin (serelaxin), recently completed Phase III clinical trials.10 Relaxin is produced and exerts its physiological effects locally in a variety of tissues but also acts in the circulatory system, especially during pregnancy.11 Interestingly, a highly sensitive preassembled signalosome capable of responding to extremely low levels of circulating relaxin was recently identified,12 and thus, there are mechanisms for sensing and responding to circulating relaxin concentrations in the sub-picomolar and nanomolar ranges. Mature relaxin consists of two peptide chains, B and A, which are held together by three disulfide © XXXX American Chemical Society

Received: November 12, 2014 Accepted: December 29, 2014

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DOI: 10.1021/cb500918v ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Human relaxin/insulin hormone family (A). Sequences of selected peptides with conserved cysteine residues highlighted and disulfide bond connectivities indicated by lines. (B) X-ray crystal structure of the H2 relaxin dimer (PDB ID 6RLX). The two H2 relaxin monomers are shown in ribbon representation in red and blue respectively, disulfide bonds in ball-and-stick, and the cysteines and terminal residues are labeled with residue numbers.

Figure 2. Chemical shift and NOE differences between H2 relaxin amide (A) and H2 relaxin (B). The displayed regions of the NOESY spectra of the two peptides highlight the differences seen for ValB16, GlnB19 and TyrA3.

to occur. RXFP1 and RXFP2 have been shown to exist as constitutive dimers having negative cooperativity in ligand binding.22 Here, we report the structural characterization of H2 relaxin using solution NMR spectroscopy and provide new mechanistic insights into its function. We show that H2 relaxin is dimeric in solution and that the negative charge on the C-terminus of the A-chain plays a critical role in self-association. Amidation of the C-termini favors a monomeric form. We also show that selfassociation of H2 relaxin does not appear to be required for structural integrity or biological activity. The amidated analogue retains full activity at the RXFP1 receptor and has a similar three-dimensional structure. Finally, the dynamic properties of the H2 relaxin structures are described using chemical shift and NMR relaxation analysis.

H2 relaxin binds with high affinity to its endogenous receptor, relaxin family peptide receptor-1 (RXFP1), as well as the INSL3 receptor, RXFP2.18 RXFP1 and RXFP2 are class A G-protein coupled receptors (GPCRs) characterized by a large ectodomain containing ten leucine-rich repeats (LRR) and a Nterminal low-density lipoprotein class A (LDL-A) module. The LDL-A module is not required for ligand binding but, interestingly, is crucial for receptor signal activation.19 The primary ligand-binding site of RXFP1/2 is located in the LRR domain and a key motif in H2 relaxin, RxxxRxxI, which is located in the B-chain is predicted to interact with the LRR.20,21 A complex RXFP1 activation model has been proposed involving multiple critical events, where the H2 relaxin Bchain binds to the LRR domain, generating a receptor conformational change, allowing interactions between the H2 relaxin A-chain, the LDL-A module and the extracellular loops B


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Figure 3. Secondary chemical shift analysis. Secondary shifts, that is, deviations of measured shifts from random coil shifts are presented for (A) 1Hα, (B) 13Cα, and (C) 13Cβ. Negative values for 1Hα and 13Cβ and positive 13Cα values indicate a helical conformation and positive 1Hα and 13Cβ values together with negative 13Cα indicate an extended β-sheet conformation.

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RESULTS AND DISCUSSION

dioxane (hydrodynamic radius 2.12 Å) as an internal standard. For a sample comprising ∼5 mM H2 relaxin, a hydrodynamic radius of 18.8 Å was observed. Based on the equation derived by Wilkins et al., Rh = (4.75 ± 1.11)N0.29±0.02 Å, where N is the

NMR Diffusion Studies. To investigate the oligomeric state of H2 relaxin translational diffusion of the peptide was measured using pulsed-field gradient NMR experiments with C


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Figure 4. Stereoview of a superposition of the 20 lowest energy structures of the H2 relaxin amide. The A-chain and B-chains are shown in light and dark blue, respectively, and the disulfide bonds are shown in yellow. Chain termini are labeled with chain identifiers and residue numbers.

number of residues and Rh the hydrodynamic radius,23 this radius is estimated to correspond to a protein of ∼115 residues, which is consistent with a H2 relaxin dimer (2 × 53 = 106 residues). A 10-fold diluted sample with a concentration of ∼0.5 mM H2 relaxin showed an apparent hydrodynamic radius of 17.0 Å. This equates to a protein of ∼81 residues; thus, even at concentrations at the lower end of what can be studied by NMR spectroscopy, a significant proportion of the peptide is dimeric. From analysis of the crystal structure it is clear that formation of the dimer results from a symmetric association of a hydrophobic interface region centered around TyrA3, ValB16, GlnB19, and IleB20. Furthermore, a pair of ionic interactions between the positive charges of ArgB13 and ArgB17 in one monomer and the negatively charged C-terminal carboxyl group of CysA24 in the second monomer, is located on the fringe of this interface. To probe whether removal of these latter interactions was sufficient to disrupt the dimerization under the studied conditions, a synthetic analogue with amidated C-termini of both the A- and B-chains was produced. This peptide was subjected to NMR diffusion analysis and was found to have a diffusion hydrodynamic radius of 15.0 or 14.6 Å at 0.35 mM or 0.7 mM, respectively. These values equate to estimates of ∼53 and ∼48 residues, which are consistent with a H2 relaxin monomer (53 residues), and thus give no indication of a concentration-dependent self-association for this analogue. NMR Resonance Assignments. 2D NMR data were recorded for both H2 relaxin and H2 relaxin amide and assigned using homonuclear sequential assignment strategies.24 For H2 relaxin at 5 mM, the quality of the data was generally good, with excellent signal dispersion and signal-to-noise. However, the spectral lines were generally broad as a result of the high molecular weight of the dimer. Additional specific broadening was observed for a number of resonances, including the CysA10 to CysA15 A-chain intra disulfide bond, the Cterminal part of the A-chain, where the amide protons of PheA23 and CysA24 were broadened beyond detection, and throughout one face of the B-chain helix. Furthermore, no resonances could be assigned to MetB4. The line-broadening around the disulfide bond is consistent with observations in other relaxins15−17 and suggests that this region undergoes conformational changes on the milli-microsecond time scale. The other regions of broadening are around the dimerization interface and are probably a result of the dimerization. H2 relaxin amide also showed excellent signal dispersion and had generally sharper lines, which allowed complete resonance assignments, with the exception of MetB4. However, line broadening was seen for CysA10 and CysA15, confirming that the dynamics of this region is not related to dimerization. Some differences in the spectral appearance of the peptides are highlighted in Figure 2. In H2 relaxin, one of the ValB16

methyl groups is unusually broadened and shifted upfield to ∼0.5 ppm. In contrast, in H2 relaxin amide this resonance is sharp and has a chemical shift of ∼0.9 ppm. Furthermore, in native H2 relaxin, both ValB16 methyl groups display characteristic NOE cross peaks to the aromatic side chain protons of TyrA3. These are intermolecular NOEs as the shortest distance between ValB16 and TyrA3 within one H2 relaxin molecule is >7 Å, whereas ValB16 and TyrA3 from the two different monomers contact each other at the dimer interface.13,14 Other notable differences include the chemical shifts and appearance of the aromatic resonances of the TyrA3 side chain. The chemical shifts of the Hδ/Hε resonances of TyrA3 are 5.85/6.46 ppm and 6.20/6.49 ppm in H2 relaxin and H2 relaxin amide, respectively. In H2 relaxin, the Hδ protons are also significantly broadened. The TyrA3 side-chains from the two molecules are in direct contact in the dimer, explaining this observation. Another key residue in the dimer interface is GlnB19. A hydrogen bond is formed between the phenolic group of TyrA3 and the GlnB19 side-chain carbonyl in the dimer. Again the chemical shifts of the side chain amide protons differ and in H2 relaxin the amide protons are significantly broadened. These shift differences and NOEs confirm the dimeric and monomeric states of the peptides under the studied conditions and provide evidence that the structure of the dimeric form of H2 relaxin in solution is fully consistent with the dimer structure observed in crystallographic studies. Chemical Shift Analysis. To utilize the highly diagnostic nature of Cα and Cβ chemical shifts for predicting local structure 1H−13C HSQC spectra at natural abundance of 13C were recorded for both H2 relaxin and H2 relaxin amide. The majority of assignments of Cα and Cβ shifts could be inferred from the 1H assignments. Figure 3 shows the secondary shifts, that is, the difference between observed chemical shifts and the corresponding reported random coil shift.25 From these data, it is clear that both the dimeric and monomeric are structurally nearly identical, and that the pattern of shifts for both peptides is thus consistent with a typical relaxin-like fold with two αhelical segments separated by a β-strand in the A-chain and one α-helical segment and a β-strand in the B-chain. All assigned chemical shifts were used as input for the program TALOS+,26 which predicts a ϕ and ψ angle combination for each residue based on patterns of secondary shifts. For the majority of resonances of both H2 relaxin and H2 relaxin amide, ϕ−ψ angle combinations could be confidently predicted by TALOS +. These angles were in excellent agreement with each other and with the observed angles in the crystal structure of the dimer of H2 relaxin, again confirming the similarities between the folds of the monomer and dimer in solution, and in the crystallographic dimer form. D


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ACS Chemical Biology 3D Structure Determination. The 2D NOESY spectrum of H2 relaxin at 5 mM concentration was crowded with too much overlap of the numerous intra- and intermolecular NOEs to resolve a full structure of the dimer. Instead, we focused on the monomeric H2 amide and determined the full NMR solution structure. Figure 4 shows a stereoview of the overlay of the 20 lowest energy structures, from which it is clear that the peptide adopts a fold that is generally well-defined. The molecular core is represented by three helical segments comprising residues A3−12, A17−23, and B12−24, as well as a small sheet comprising two strands involving residues A15− 16 and B7−8. These elements of secondary structure are held together by the disulfide bonds and a hydrophobic core, which includes the side chains of residues TyrA3, LeuA6, CysA10, CysA15, LysA17, LeuA20, PheA23, LeuB15, AlaB18, GlnB19, AlaB21, and IleB22. In addition, the charged portion of the LysA17 side chain forms a salt bridge with GluB6. The structure is more disordered around the chain termini, which is common for relaxins and other proteins in which the termini are not restrained by stabilizing features such as disulfide bonds. The energies and structural statistics for the solution structure of H2 relaxin amide are given in Table 1 and highlight that the structural models are in good agreement with experimental data and have good covalent geometries. H2 Relaxin Monomeric and Dimeric Structures. Figure 5A shows an overlay of the solution structure of H2 relaxin amide with the H2 relaxin crystal structure. Although the structures are very similar, some differences are seen, particularly around the B-chain termini. In the crystal structure residues B2−B4 form a 310 helical turn that is packed against the C-terminal part of the B-chain. There are indications of a turn also in solution but this region appears more flexible with MetB4 not visible due to broadening. The length of the B-chain helix and the orientation of the B-chain C-terminus also differ, but again, this region appears to be dynamic in solution, and also, in the crystal analysis the terminal residues lack electron density.13 Other differences include the orientation of the key ArgB13-ArgB17 side chains and slight differences in the orientation of the A-chain helices. The conformations of the Arg side chains in the monomer are not well-defined, as expected for charged surface-exposed residues. In contrast, in the crystal structure dimer these side chains are ordered to form the salt-bridge interactions with the A-chain C-terminus, and this interaction may also cause a perturbation of the orientation of the C-terminal A-chain helix. H2 relaxin was confirmed to be fully dimeric in solution at ∼5 mM concentration by NMR diffusion measurements. The spectra revealed only one set of resonances, confirming a symmetrical dimer with each residue being in an identical environment in the monomer units. Considerable evidence suggests the arrangement of the dimer is identical to that seen in the crystal structure. Key interactions around the dimer interface are illustrated in Figure 5B and include interactions between the TyrA3, ValB16, GlnB19, and IleB20 side-chains, as well as the ionic interaction between ArgB13, ArgB17, and the A-chain C-termini. Distinct differences in chemical shifts and line widths were observed for these residues, consistent with the dimer arrangement observed in the crystal structure. Functional and Serum Stability Assays. To investigate whether removal of the negative charges of the two C-termini and disruption of the ability to self-associate influences the bioactivity of H2 relaxin, we compared the ability of H2 relaxin amide to bind and induce activation of RXFP1 to that of native

Table 1. NMR Distance and Dihedral Statistics and Structure Statistics NMR Distance and Dihedral Statistics distance constraints total inter-residue NOE sequential medium range long range hydrogen bonds

(|i − j| = 1) (|i − j| ≤ 4) (|i − j| ≥ 5) (2 per H-bond) dihedral angles

ϕ ψ χ1

204 78 67 30 45 35 9

Structure Statistics violations distance constraints (>0.3 Å) 3 (/ 20 structures) dihedral angle constraints (>3°) 0 max distance constraints violation (Å) 0.3 max dihedral angle constraints (deg) 2.8 energies (kcal/mol, mean ± standard deviation) overall −1786.1 ± 35.6 bond 23.84 ± 2.03 angles 79.64 ± 9.23 improper 25.85 ± 3.06 vdw −207.9 ± 10.65 NOE (experimental) 0.377 ± 0.056 cDih (experimental) 1.55 ± 0.698 dihed 252.0 ± 3.88 elec −1961.4 ± 29.3 deviation from idealized geometry bond length (Å) 0.0113 ± 0.00058 bond angles (deg) 1.3445 ± 0.07371 impropers (deg) 1.2918 ± 0.10689 avg. pairwise root-mean-square deviationa (Å) backbone atoms 0.71 ± 0.19 heavy atoms 1.74 ± 0.31 Ramachandran statisticsb most favored regions (%) 89.1 additionally allowed (%) 8.7 generously allowed (%) 2.2 disallowed (%) 0 a

Pairwise root-mean-square deviation was calculated from 20 refined structures over amino acids A1−24 and B5−24. bFrom Procheck version 3.5.4.

H2 relaxin.19 Both binding and activation were indistinguishable in cell based assays (Figure 6). The peptides show activity in the nanomolar range and based on the association constant from ultracentrifugation experiments in solution, H2 relaxin is not expected to be dimeric at these concentrations. However, the environment in the assays, with the presence of receptors and other membrane components, is significantly different from an aqueous buffer. Under these conditions, the dimeric species could play a role, but the fact that H2 relaxin amide retains full function strongly suggests that the active state of the peptide is the monomeric form. It can further be concluded that neither of the negatively charged C-terminal carboxyl groups are important for the receptor interaction, unlike in some other members of the relaxin family, including INSL5.27 The half-life of H2 relaxin amide and H2 relaxin in serum was also determined (Figure 6) and although the amidated form E


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Figure 5. (A) Comparison of the H2 relaxin amide monomer (red with orange disulfides) and the crystal structure of H2 relaxin (blue with yellow disulfides). Residues ArgB13, ArgB17, and IleB20, which are involved in the binding interaction to RXFP1, are shown in stick representation. (B) Key features of the H2 relaxin dimer interface. ArgB13 and ArgB17 are involved in salt bridge interactions to the carboxylate group of CysA24 across the crystal dimer interface. Disulfide bonds are shown in yellow.

Figure 6. Biological activity and serum stability. (A) Competition binding of H2 relaxin amide in comparison to recombinant H2 relaxin in HEK-293T cells stably transfected with RXFP1using Eu−H2 relaxin expressed as % specific binding. (B) cAMP activity responses expressed as the percentage of the maximal H2 relaxin response. (C) In vitro serum stability presented as % peptide remaining in human serum over time. Symbols represent the means, and the vertical bars SEM (standard error of the mean) of three independent experiments.

appeared to be slightly more stable (3.4 h vs 2.5 h), there was no significant difference (p < 0.05). How Does H2 Relaxin Engage RXFP1? The RxxxRxxI motif in the H2 relaxin B-chain helix has long been known to be important for bioactivity.20 Similar motifs are found in other members of the relaxin family and are invariably involved in receptor interactions.15,28 The H2 relaxin receptor RXFP1 and the closely related INSL3 receptor RXFP2 are large GPCRs, with the extracellular N-terminal region comprising both an LDL-A module and a LRR domain, and require a complex mechanism of activation. The B-chain RxxxRxxI motif appears to target a primary binding site located in the extracellular LRR domain, but additional interactions are also required. Truncation studies on INSL3 have revealed that these additional interactions include the N-terminal portion of the A-chain, with truncations of nine residues yielding a high affinity antagonist.29 For H2 relaxin, however, despite several dozen analogues having been made, including truncated and Ala substituted variants,30,31 not a single analogue that retains high affinity binding but loses the ability to activate the receptor has been found. Instead, a loss of activation is always a result of

a loss in binding, highlighting that, for this, peptide binding and activation are intimately linked.30 Studies on chimeric relaxins with a H2 relaxin B-chain and Achains from other members of the family confirm the importance of both the A-chain and B-chain of H2 relaxin for interaction with RXFP1 and RXFP2.32 Ala replacements of residues in the A-chain have suggested that no single amino acid is the driver of a secondary interaction but have identified contributions from TyrA3, LeuA20, and PheA23.33 TyrA3 and PheA23 are partly exposed in the H2 relaxin monomer structure whereas LeuA20 is fully buried in the hydrophobic core, suggesting that the latter residue might play a structural role, whereas the former residues are able to engage the receptor. This interaction probably involves the extracellular loops of RXFP1, thereby providing the coordination between the LRR domain and the transmembrane region required for repositioning of the LDL-A module and activation of the receptor. The fact that it is not possible to ablate the secondary binding site while retaining high affinity antagonistic binding through the primary binding site in the LRR33 suggests that this secondary binding contributes substantially to the overall affinity of H2 relaxin. This is consistent with the secondary F


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Figure 7. 13C NMR relaxation data for the H2 relaxin. Heteronuclear 1H−13C NOE data are shown as mean ± standard deviations from three repeated experiments (A). T1 data are shown as best fit values with error bars representing the 95% confidence intervals of the fits (B). Predicted order parameters of the H2 relaxin dimer and H2 relaxin amide monomer derived from the TALOS+ analysis (C).

and 0.76, respectively. However, distinct increases and decreases of the NOE and T1 values toward ∼1.6−1.8 and ∼0.5 s, respectively, are seen for the terminal few residues of the A-chain N-terminus and the B-chain N- and C-termini, consistent with significant fast internal motion in these regions. Dynamic regions can also be predicted based on chemical shifts,34 as analyzed by the TALOS+ program.26 Figure 7C shows the TALOS+ predicted S2 parameters, which theoretically range from 0 for unrestricted internal motion to 1 for complete absence of internal motion. The predictions are generally consistent with the experimental relaxation data, highlighting a rigid conformation in the central part of the chains with increased flexibility in terminal regions, in particular, at the B-chain C-terminus. Both peptides have similar values, showing that the dynamics of the fold is not significantly affected by dimer formation, although there is a trend of higher S2 values in the B-chain helix of H2 relaxin and at the end of the C-terminal helix of the A-chain, suggesting that the fold is slightly stabilized in the dimer. Is the Flexibility Linked to Activity? Structural flexibility in proteins is often linked to function; for example, structural rearrangements can be required for enzymes to allow substrates

interaction involving a number of residues spread across the Achain. 13 C NMR Relaxation Analysis. NMR studies of relaxins, including H3 relaxin, INSL3, and INSL5, have all suggested that the relaxin fold is dynamic, with regions of the structures being associated with varying degrees of line broadening.15−17 During our studies of H2 relaxin, similar observations were made, with regions of line broadening being identified around the CysA10−CysA15 disulfide bond in particular. To further investigate the intrinsic dynamics of H2 relaxin, we measured 13 C relaxation data for H2 relaxin at natural abundance. Figure 7 shows the T1 relaxation values and steady state heteronuclear 1 H−13C NOEs of H2 relaxin measured at 600 MHz. Unlike line-broadening phenomena, which are sensitive to dynamic fluctuations on the milli−microsecond time scale, relaxation parameters are sensitive to motion on the pico−nano second time scale. From Figure 7 it is clear that in the H2 relaxin dimer the majority of residues in the molecular core have 1H−13C NOE values of ∼1.2 and T1 values of ∼0.75 s. These values are consistent with a well-ordered relatively rigid fold, with the theoretical values for a predicted order parameter of ∼0.8 and a predicted correlation time for the dimer of ∼5 ns being 1.16 G


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secretory granules when the peptide is present in very high concentration. Insulin is stored as Ca2+ and Zn2+ stabilized hexamers, which is a critical feature of insulin pharmacodynamics, as monomers have a tendency to aggregate into toxic fibrils.37 Several mutations of insulin that make its overall structure less stable increase activity due to an increased ability to adopt an active conformation,38 but increased flexibility can also increase toxic fibril formation.35 The H2 relaxin dimer might play a similar role; however, this would have to be further investigated, particularly in relation to differences in aggregation between relaxins from different species. Predicted order parameters for H2 relaxin and H2 relaxin amide do suggest an increased ordering of both the A-chain C-terminal helix and the B-chain helix, when in a dimer form (Figure 7), which would be consistent with the dimer stabilizing the folded structure. Conclusion. In this study, we have characterized the structural features and solution dynamics of H2 relaxin and its synthetic amidated analogue, H2 relaxin amide. These peptides are equipotent at the RXFP1 receptor and are structurally very similar, but the amide variant does not selfassociate into the characteristic dimers seen in the crystal structure and in solution for native H2 relaxin. This confirms that the monomeric state is the likely pharmacologically active form of H2 relaxin, and that unlike several other members of the family, a C-terminal negative charge is not required for activity. The fold of H2 relaxin is dynamic both on fast and slow time scales, which may contribute to function by allowing optimization of the extensive receptor binding surface.

to enter or leave active sites, and ligands may require conformational freedom for binding to receptor targets, which, in turn, alters the receptor’s conformation to an active form. H3 relaxin, INSL3, and INSL5 all have been shown to be rather dynamic in solution, as evident from selective linebroadening of residues in NMR spectra.15−17 Here, we show that H2 relaxin, both in its native dimer form and as an engineered monomer, also shows severe signal broadening in distinct regions of their structure. These include a number of residues around the A-chain internal disulfide bond, and in addition, flexibility around the termini is evident from analysis of line shapes, chemical shifts, and NMR relaxation data. Flexibility, and its link to function, has been intensively characterized in H2 relaxin’s close relative, insulin. Significant structural changes in the conformation of the B-chain termini in insulin are required for receptor binding. The A-chain Nterminal helix, carrying several critical residues including IleA2 and ValA3, has also been shown by 13C chemical shifts to be more flexible,35 which may favor the correct positioning of these residues in the complex. A recent crystal structure of insulin in complex with its receptor reveals numerous contacts involving the B-chain helix and both the A-chain helices.36 The binding of H2 relaxin to its receptor is less well characterized. However, it appears that H2 relaxin also utilizes an extensive binding surface spanning several regions of the peptide, which might also require optimization of its conformation upon binding. The most dynamic regions of H2 relaxin based on predicted order parameters and experimental relaxation data are the B-chain N- and C-termini. Truncation studies show that neither of these regions are essential for binding.31 However, there is also evidence for flexibility around the A-chain termini, which appears to be involved in receptor binding. The intra-Achain disulfide bond is positioned at the bottom of the ‘U’ shaped A-chain, and thus, the conformational flexibility seen here could function as a “hinge” allowing repositioning of the two helices for optimal binding. What Is the Role of the Relaxin Dimer? The ability of H2 relaxin to self-associate into well-defined dimers is intriguing. The dimerization constant for H2 relaxin in 10 mM citrate buffer, pH 5, is in the low micromolar range.14 Thus, the fact that the peptide shows bioactivity at substantially lower concentrations would perhaps suggest that it binds to RXFP1 as a monomer. However, the environment in which H2 relaxin exerts its activity is highly complex. RXFP1 forms dimers,22 thus multiple high affinity receptor binding sites, as well as other membrane components, are present within close proximity. Conformational changes as a result of interactions with receptors may increase propensity for self-association and with a high local concentration of peptide, dimerization could play a role in coordination of the structural rearrangement required for receptor activation. The dimer interface involves a number of residues shown to be important for bioactivity, including the ArgB13, ArgB17, and IleB20 cassette, as well as TyrA3. This could be interpreted as suggesting the dimer is critical for function and that the importance of these residues is a result of their role in the formation of the dimer. We show, however, here that destabilization of this dimer form through amidation of the C-terminal groups does not affect bioactivity. Thus, the dimer is unlikely to be a bioactive species, and these residues represent true “hot spots” for receptor interactions. It is instead interesting to speculate that the formation of dimers might have evolved to stabilize relaxin to prevent toxic unfolding and aggregation during biosynthesis and storage in

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METHODS

Peptides. Native recombinant H2 relaxin (serelaxin) was kindly provided by Corthera Inc. H2 relaxin amide (in which both the A and B-chain C-termini were amidated) was synthesized using Fmoc chemistry and regioselective disulfide bond formation as previously described for a range of relaxin peptides.39,40 The peptide was purified and characterized by RP-HPLC and MALDI-TOF MS. NMR Spectroscopy. Diffusion Measurements: Samples of various peptide concentrations in 100% D2O (pH ∼ 4 not corrected for isotope effects) were investigated by pulsed-field gradient diffusion experiments with a pseudo-two-dimensional (2D) sequence using stimulated echo longitudinal encode−decode.41 The strength of the gradient was varied between 2% and 95% over 40 spectral data points with all delays held constant. Diffusion delays were optimized for each sample to give an attenuation of the signals by ∼90%. 1,4-Dioxane was used as an internal standard with known diffusion properties and added to each sample to a final concentration of 0.01%. The diffusion data were analyzed using the DOSY toolbox.42 Resonance Assignments: Spectral data for resonance assignments included 2D 1H−1H TOCSY (mixing time 80 ms), NOESY (mixing time 100 or 200 ms), and DQF-COSY recorded at either 600 or 900 MHz on Bruker Avance or Avance II spectrometers, respectively. Temperatures were varied in the range 283−308 K to resolve ambiguities due to overlapping amide proton chemical shifts. Chemical shifts were referenced to internal 2,2-dimethyl-2-silapentanesulfonic acid (DSS) at 0.00 ppm. Natural Abundance 13C NMR: 1H−13C HSQC spectra were recorded on samples of 1−5 mM of H2 relaxin or 0.7 mM H2 relaxin amide dissolved in 100% D2O at 298 K. 13C NMR relaxation data, including steady state heteronuclear NOEs and T1 were measured for the Cα nuclei at 600 MHz, using HSQC based experiments on a sample of 5 mM H2 relaxin. All data were recorded with 128 scans and a relaxation delay between scans of 5 s. For T1 determination, 11 data points were recorded with relaxation delays of 0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, and 3.0 s. Relaxation data were fitted to exponential decay curves, from which the relaxation H


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ACS Chemical Biology constant R1(=1/T1) was extracted, using the program Prism 5.0 (GraphPad). Structure Determination H2 Relaxin Amide. For the structure determination of H2 relaxin amide structural constraints, including interproton distances, backbone and side-chain dihedral angles, and hydrogen bonds, restraints were derived from NMR spectroscopy data. Distance restraints were derived from peak intensities in NOESY spectra recorded with a mixing time of 100 ms at 600 MHz, 298 K, and pH ∼ 4 on 0.7 mM samples in either 90% H2O/10% D2O or 100% D2O. The NOESY data were analyzed, and peak volumes were integrated in the program CARA and translated into distance restraints with appropriate pseudoatom corrections using the program CYANA 2.0.43 Backbone ϕ and ψ dihedral angle restraints were derived from a TALOS+ analysis of the HN, Hα, Cα, and Cβ chemical shifts.26 Additional ϕ restraints of −100 ± 80° were used for residues where no prediction was made by TALOS+, but a positive ϕ angle could be ruled out based on a strong sequential Hαi−1-HNi NOE in comparison to the intraresidual Hαi-HNi NOE. Side chain χ1 dihedral angle restraints and stereospecific assignments of Hβ methylene pairs were derived based on analysis of 3JHαHβ coupling constants and intraresidual NOE patterns. Hydrogen bonds were identified by analysis of amide temperature coefficients based on TOCSY spectra recorded in the temperature range 283−308 K. ΔδHN/ΔT values > −4.6 ppb/K are indicative of hydrogen bonds.44 For amides where suitable hydrogen bond acceptors could be confidently identified in preliminary structures, hydrogen bonds were included as restraints in the final round of structure calculations. The final set of structures of H2 relaxin amide was generated using methods described previously.45 Briefly, the structural restraints were included in simulated annealing and molecular dynamics simulations within the program CNS.46 A set of 50 structures was generated by torsion angle dynamics and subsequently refined and energy minimized in a shell of explicit water. The 20 lowest energy structures with good covalent geometry and consistency with experimental data were chosen to represent the solution structure of H2 relaxin amide. Cell Based RXFP1 Assays. The ability of native H2 relaxin and H2 relaxin amide to bind to47 and activate the RXFP1 receptor19 was compared as previously described. Data points were measured in triplicate, and each experiment was repeated five times. Serum Stability Assays. Human male serum was preincubated for 15 min at 37 °C before H2 amide or H2 acid (5 μg peptide per 95 μL serum) was added to the serum. Aliquots (100 μL) were taken out at different time points and 900 μL ammonium acetate, pH 3, were added to each sample before incubation on ice for 30 min. The peptide was extracted from the serum using an Oasis HLB 3 cc 60 mg cartridge (Waters). The peptide eluate was lyophilized and redissolved in 1% formic acid before being analyzed by LC-MS. The serum stability data was analyzed using one-phase exponential decay equation followed by a t test (Prism 6).

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Fellows. R.A.D.B. is a National and Medical Health Research Council (NHMRC, Australia) Senior Research Fellow. J.D.W. is an NHMRC Principal Research Fellow. D.J.C. is an NHMRC Senior Principal Research Fellow. This work was supported by the Faculty of Natural Sciences and Technology, Linnaeus University (K.J.R.) and by NHMRC Project grants 1023078 (J.D.W., R.A.D.B., M.A.H.) and 1043750 (R.A.D.B., K.J.R.).

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(1) Hisaw, F. L. (1926) Experimental relaxation of the pubic ligament of the guinea pig. Proc. Soc. Exp. Biol. Med. 23, 661−663. (2) Unemori, E. N., Pickford, L. B., Salles, A. L., Piercy, C. E., Grove, B. H., Erikson, M. E., and Amento, E. P. (1996) Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo. J. Clin. Invest. 98, 2739−2745. (3) Royce, S. G., Miao, Y. R., Lee, M., Samuel, C. S., Tregear, G. W., and Tang, M. L. (2009) Relaxin reverses airway remodeling and airway dysfunction in allergic airways disease. Endocrinology 150, 2692−2699. (4) Unemori, E. N., Lewis, M., Constant, J., Arnold, G., Grove, B. H., Normand, J., Deshpande, U., Salles, A., Pickford, L. B., Erikson, M. E., Hunt, T. K., and Huang, X. (2000) Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites. Wound Repair Regeneration 8, 361−370. (5) Formigli, L., Francini, F., Nistri, S., Margheri, M., Luciani, G., Naro, F., Silvertown, J. D., Orlandini, S. Z., Meacci, E., and Bani, D. (2009) Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures. J. Mol. Cell. Cardiol. 47, 335−345. (6) Feng, S., Agoulnik, I. U., Bogatcheva, N. V., Kamat, A. A., KwabiAddo, B., Li, R., Ayala, G., Ittmann, M. M., and Agoulnik, A. I. (2007) Relaxin promotes prostate cancer progression. Clin. Cancer Res. 13, 1695−1702. (7) Goldsmith, L. T., Weiss, G., Palejwala, S., Plant, T. M., Wojtczuk, A., Lambert, W. C., Ammur, N., Heller, D., Skurnick, J. H., Edwards, D., and Cole, D. M. (2004) Relaxin regulation of endometrial structure and function in the rhesus monkey. Proc. Natl. Acad. Sci. U.S.A. 101, 4685−4689. (8) Samuel, C. S., Hewitson, T. D., Unemori, E. N., and Tang, M. L. (2007) Drugs of the future: The hormone relaxin. Cell. Mol. Life Sci. 64, 1539−1557. (9) van der Westhuizen, E. T., Halls, M. L., Samuel, C. S., Bathgate, R. A. D., Unemori, E. N., Sutton, S. W., and Summers, R. J. (2008) Relaxin family peptide receptorsFrom orphans to therapeutic targets. Drug Discovery Today 13, 640−651. (10) Teerlink, J. R., Cotter, G., Davison, B. A., Felker, G. M., Filippatos, G., Greenberg, B. H., Ponikowski, P., Unemori, E., Voors, A. A., Adams, K. F., Jr., Dorobantu, M. I., Grinfeld, L. R., Jondeau, G., Marmor, A., Masip, J., Pang, P. S., Werdan, K., Teichman, S. L., Trapani, A., Bush, C. A., Saini, R., Schumacher, C., Severin, T. M., and Metra, M. (2013) Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): A randomised, placebo-controlled trial. Lancet 381, 29−39. (11) Samuel, C. S., Du, X. J., Bathgate, R. A. D., and Summers, R. J. (2006) ‘Relaxin’ the stiffened heart and arteries: The therapeutic potential for relaxin in the treatment of cardiovascular disease. Pharmacol. Ther. 112, 529−552. (12) Halls, M. L., and Cooper, D. M. (2010) Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex. EMBO J. 29, 2772−2787. (13) Eigenbrot, C., Randal, M., Quan, C., Burnier, J., O’Connell, L., Rinderknecht, E., and Kossiakoff, A. A. (1991) X-ray structure of human relaxin at 1.5 A. Comparison to insulin and implications for receptor binding determinants. J. Mol. Biol. 221, 15−21. (14) Shire, S. J., Holladay, L. A., and Rinderknecht, E. (1991) Selfassociation of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism. Biochemistry 30, 7703− 7711.

ASSOCIATED CONTENT

Accession Codes

The structure and chemical shifts have been submitted to the PDB and BMRB databases and given the accession numbers 2mv1 and 25238, respectively.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +61 7 33651403. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the use of the Queensland NMR Network 900 MHz spectrometer. L.M.H.K. is a recipient of a Swedish Research Council Post-Doctoral Fellowship. N.L.D. and K.J.R. are Australian Research Council (ARC) Future I


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Solution Structure, Aggregation Behavior, and Flexibility of Human

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