Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m511210200
ISSN1083-351X
AutoresK. Johan Rosengren, Feng Lin, Ross A. D. Bathgate, Geoffrey W. Tregear, Norelle L. Daly, John D. Wade, David J. Craik,
Tópico(s)Pregnancy-related medical research
ResumoRelaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing TrpB27 to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135. Relaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing TrpB27 to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135. Recent developments have caused considerable excitement in the relaxin field, including the discovery of a new member of the relaxin family, relaxin-3 (1Bathgate R.A.D. Samuel C.S. Burazin T.C. Layfield S. Claasz A.A. Reytomas I.G. Dawson N.F. Zhao C. Bond C. Summers R.J. Parry L.J. Wade J.D. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), and the identification of several long sought after relaxin receptors (2Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (686) Google Scholar). Prior to the discovery of the relaxin-3 gene (RLN3), only one relaxin gene had been characterized in most mammals, with the exception of humans and higher primates, in which two separate genes, RLN1 and RLN2, were known (3Hudson P. Haley J. John M. Cronk M. Crawford R. Haralambidis J. Tregear G. Shine J. Niall H. Nature. 1983; 301: 628-631Crossref PubMed Scopus (169) Google Scholar, 4Hudson P. John M. Crawford R. Haralambidis J. Scanlon D. Gorman J. Tregear G. Shine J. Niall H. EMBO J. 1984; 3: 2333-2339Crossref PubMed Scopus (164) Google Scholar). The product of the human RLN2 gene, human relaxin-2 (H2 relaxin), is the functional ortholog of the RLN1 gene product from non-primate species, which is termed relaxin. The function of the product of the human RLN1 gene, human relaxin-1 (H1 relaxin), is unknown; and indeed, a native H1 relaxin peptide has not been isolated (5Wilkinson T.N. Speed T.P. Tregear G.W. Bathgate R.A.D. BMC Evolutionary Biology. 2005; http://www.biomedcentral.com/1471-2148/5/14PubMed Google Scholar). Hence, throughout this work, "relaxin" will refer to the pregnancy hormones H2 relaxin and non-primate relaxin. Relaxin-3 is the ancestor of the entire relaxin peptide family (5Wilkinson T.N. Speed T.P. Tregear G.W. Bathgate R.A.D. BMC Evolutionary Biology. 2005; http://www.biomedcentral.com/1471-2148/5/14PubMed Google Scholar), and RLN3 genes have been identified in all mammalian genomes as well as in the genomes of chicken, frog, and various fish species. In contrast, the RLN1 gene is found only in mammals. Fig. 1 shows a sequence comparison of the product of the human RLN3 gene, human relaxin-3 (H3 relaxin); H2 and H1 relaxins; and relaxin-3 orthologs from other species. Interestingly, the relaxin-3 sequences are highly conserved between species, in contrast to relaxin, which shows considerable sequence variation. Relaxin has long been regarded as a hormone mainly associated with pregnancy. Produced in the corpus luteum and/or placenta of most mammals, it has numerous pregnancy-specific actions, including the remodeling of the reproductive tract and preparation of the mammary apparatus for lactation (6Sherwood O.D. The Physiology of Reproduction.in: Knobil E. Neill J.D. Raven Press, Ltd., New York1994: 861-1009Google Scholar). However, relaxin also has important physiological roles outside of pregnancy. It inhibits collagen biosynthesis and promotes collagen breakdown (7Williams E.J. Benyon R.C. Trim N. Hadwin R. Grove B.H. Arthur M.J. Unemori E.N. Iredale J.P. Gut. 2001; 49: 577-583Crossref PubMed Scopus (167) Google Scholar, 8Unemori E.N. Amento E.P. J. Biol. Chem. 1990; 265: 10681-10685Abstract Full Text PDF PubMed Google Scholar, 9Garber S.L. Mirochnik Y. Brecklin C.S. Unemori E.N. Singh A.K. Slobodskoy L. Grove B.H. Arruda J.A. Dunea G. Kidney Int. 2001; 59: 876-882Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and causes vasodilatation in various tissues (10Bani D. Gen. Pharmacol. 1997; 28: 13-22Crossref PubMed Scopus (205) Google Scholar). Interestingly, the expression pattern of relaxin-3 differs significantly from that of relaxin, suggesting a distinctly different physiological role. The highest expression of relaxin-3 in all species examined to date, including humans, is in the brain. In rats (11Burazin T.C. Bathgate R.A.D. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Crossref PubMed Scopus (175) Google Scholar) and mice (1Bathgate R.A.D. Samuel C.S. Burazin T.C. Layfield S. Claasz A.A. Reytomas I.G. Dawson N.F. Zhao C. Bond C. Summers R.J. Parry L.J. Wade J.D. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), the expression is localized to a specific region of the dorsal tegmental region of the pons called the nucleus incertus. Anatomical studies suggest that this nucleus is involved in a midbrain behavior control network that influences circuits regulating locomotion, attention, and learning processes and that responds to stress-related neuroendocrine signals (12Goto M. Swanson L.W. Canteras N.S. J. Comp. Neurol. 2001; 438: 86-122Crossref PubMed Scopus (189) Google Scholar). Mapping studies have demonstrated that relaxin-3-immunoreactive nerve fibers emanating from the nucleus incertus innervate numerous regions of the brain, suggesting that the nucleus incertus utilizes relaxin-3 as a neurotransmitter (13Tanaka M. Iijima N. Miyamoto Y. Fukusumi S. Itoh Y. Ozawa H. Ibata Y. Eur. J. Neurosci. 2005; 21: 1659-1670Crossref PubMed Scopus (195) Google Scholar). These data, coupled with the remarkable conservation of the relaxin-3 peptide from fish to humans, suggest that relaxin-3 has highly important, but presently unknown, central actions. Indeed, a very recent study suggests that relaxin-3 is involved in appetite regulation (14McGowan B.M. Stanley S.A. Smith K.L. White N.E. Connolly M.M. Thompson E.L. Gardiner J.V. Murphy K.G. Ghatei M.A. Bloom S.R. Endocrinology. 2005; 146: 3295-3300Crossref PubMed Scopus (150) Google Scholar), and another study has demonstrated that relaxin-3 mRNA production is increased in response to water-restraint stress (13Tanaka M. Iijima N. Miyamoto Y. Fukusumi S. Itoh Y. Ozawa H. Ibata Y. Eur. J. Neurosci. 2005; 21: 1659-1670Crossref PubMed Scopus (195) Google Scholar), suggesting a role in the stress response. Members of the relaxin family are structurally similar to insulin, comprising two peptide chains that are linked by two disulfide bonds, with the A-chain containing a third, intrachain disulfide bond. However, unlike insulin, whose receptor is a tyrosine kinase, members of the relaxin peptide family bind to G-protein-coupled receptors (GPCRs). 2The abbreviations used are: GPCRs, G-protein-coupled receptors; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPLC, high pressure liquid chromatography; NOE, nuclear Overhauser effect. The first receptors identified responding to relaxin stimuli were the leucine-rich repeat-containing GPCRs LGR7 and LGR8 (2Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (686) Google Scholar), which both have low nanomolar affinity for relaxin and which are capable of mediating the actions of relaxin through a cAMP-dependent pathway (2Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (686) Google Scholar). Interestingly, relaxin-3 also activates LGR7, albeit with a lower affinity than relaxin, but has a significantly reduced affinity for LGR8 (15Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A.D. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Recent work has identified two additional orphan GPCRs, GPCR135 and GPCR142, which also respond to relaxin-3 activation (16Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 17Liu C. Chen J. Sutton S. Roland B. Kuei C. Farmer N. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50765-50770Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). On the basis of the higher affinity of relaxin-3 for GPCR135 and their coexpression in regions of the brain, it was concluded that relaxin-3 is the endogenous ligand for GPCR135, whereas the primary ligands for LGR8 and GPCR142 are two other members of the relaxin peptide family, INSL3 (insulin-like peptide-3 or Leydig insulin-like peptide) (18Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A.D. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar) and INSL5 (insulin-like peptide-5) (19Liu C. Kuei C. Sutton S. Chen J. Bonaventure P. Wu J. Nepomuceno D. Kamme F. Tran D.T. Zhu J. Wilkinson T. Bathgate R. Eriste E. Sillard R. Lovenberg T.W. J. Biol. Chem. 2005; 280: 292-300Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), respectively. The cross-reactivity of these hormone-receptor signaling systems has raised questions about which factors are responsible for the selectivity of these ligand-receptor pairings. Moreover, as relaxin-3, GPCR135, and LGR7 are all expressed in the brain, understanding the significance of the interaction of relaxin-3 with LGR7 will be crucial in determining its function. In this study, we provide a crucial piece of the puzzle by presenting the solution structure of the human form of the most recently discovered member of this hormone family, relaxin-3. The structure reveals a relaxin/insulin fold, but with an unusual conformation of the C-terminal region of the B-chain. The structural data provide insights into the receptor specificities of this new member of the relaxin peptide family. Peptide Synthesis—The A- and B-chains were assembled as C-terminal peptide acids or amides by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase synthesis on a hydroxymethylphenoxyacetyl- or 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeryl-polyethylene glycol/polystyrene support using the following selective cysteine S-protection: Cys A10/A15 trityl; CysA11/B11, acetamidomethyl; CysA24, t-butyl; and CysB22, trityl. Following conventional trifluoroacetic acid cleavage in the presence of scavengers, the intramolecular disulfide bond of the A-chain was formed by aeration, and CysA24 (t-butyl) was displaced with S-pyridinyl by reaction with 2,2′-dipyridyl disulfide in trifluoromethanesulfonic acid. Combination of the peptide with the B-chain occurred by thiolysis, after which the third and final disulfide bond between A11 and B10 was formed by iodolysis. The purity of the peptide was confirmed by MALDI-TOF mass spectrometry (for example, peptide acid: theory, 5500.5; and found, 5499.6) and analytical reversed-phase HPLC. Biological Activity Assays—H3 relaxin acid and amide were tested for their ability to activate LGR7, LGR8, and GPCR135. Stably transfected human embryonic kidney 293T cells expressing human LGR7 or LGR8 (15Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A.D. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) and Chinese hamster ovary K1 cells stably expressing human GPCR135 (20van der Westhuizen E.T. Sexton P.M. Bathgate R.A.D. Summers R.J. Ann. N. Y. Acad. Sci. 2005; 1041: 332-337Crossref PubMed Scopus (31) Google Scholar) were stimulated for 30 min with various concentrations of H3 relaxin acid or amide. LGR7 and LGR8 were tested in parallel with H2 relaxin and human INSL3, respectively (both at 100 nm), and GPCR135 with forskolin (5 μm) to determine maximum cellular cAMP response. cAMP accumulation was measured in cell lysates as described previously (15Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A.D. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). NMR Spectroscopy—Pulsed-field gradient NMR diffusion experiments were performed with a two-dimensional sequence using stimulated echo longitudinal encode-decode (21Altieri A.S. Hinton D.P. Byrd R.A. J. Am. Chem. Soc. 1995; 117: 7566-7567Crossref Scopus (442) Google Scholar). The lengths of all pulses and delays were held constant, and 32 spectra were acquired with the strength of the diffusion gradient varying between 2 and 95% of its maximum value. The lengths of the diffusion gradient and the stimulated echo were optimized for each sample to give a total decay in the protein signal of ∼90%. Dioxane was added to the samples to a final concentration of 0.2 mm and used as an internal standard (22Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (830) Google Scholar). Samples prepared for structure determination contained ∼1 mm peptide dissolved in 90% H2O and 10 or 100% (v/v) D2O at pH 3.0. Spectra were recorded at 290, 298, and 303 K on a Bruker Avance 600-MHz spectrometer or on a Bruker DMX 750-MHz spectrometer. Two-dimensional experiments recorded included double-quantum filtered COSY; total correlation spectroscopy using an MLEV-17 spin lock sequence with a mixing time of 80 ms; and nuclear Overhauser effect (NOE) correlation spectroscopy with mixing times of 100, 150, and 200 ms. Spectra were generally acquired with 4096 complex data points in F2 and 512 increments in the F1 dimension over a spectral width of 14 ppm. Slowly exchanging NH protons were detected by acquiring a series of one-dimensional and total correlation spectra of the fully protonated peptide immediately after dissolution in D2O. As most amides disappeared within the first 2 h, resonances still visible after 2 h were considered to be protected from the solvent by hydrogen bonding. Spectra were processed on a Silicon Graphics Octane workstation using XWIN-NMR (Bruker BioSpin Corp.). The F1 dimension was generally zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied before Fourier transformation. Chemical shifts were referenced to 2,2-dimethyl-2-silapentanesulfonic acid at 0.00 ppm. Distance restraints were derived primarily from a 100-ms NOE correlation spectrum recorded at 298 K and 600 MHz. Cross-peaks were assigned and integrated in XEASY and converted to distance restraints using CYANA. Distance restraints for cross-peaks that could not be unambiguously assigned were introduced into structure calculations as ambiguous restraints. Backbone dihedral restraints were inferred from 3JNH-Hα coupling constants. Because of the generally broad lines of many amide protons, coupling constants were, in some cases, estimated from a combination of apparent coupling constants, peak intensities, and consistency with preliminary structures. The dihedral angle φ was restrained to -120 ± 40° for 3JNH-Hα > 8 Hz and to -60 ± 30° for 3JNH-Hα < 5 Hz. Additional φ angle restraints of -100 ± 80° were included where a positive angle could be excluded based on a strong sequential Hαi-1-HNi NOE compared with the intraresidue Hαi-HNi NOE. Side chain χ1 angles and stereospecific assignments were determined on the basis of observed NOE and 3JHα-Hβ coupling patterns. For a predicted t2 g3 side chain conformation, the χ1 were restrained to -60 ± 30°(TrpA13residues,GluA19, CysA24, ArgB8, LeuB9, CysB10, and CysB22); and for a g2t3 conformation, the angles were constrained to 180 ± 30° (residues LysA12, LysA17, LeuA23, PheB14, and ArgB26). No residues could be confirmed to be in the g2 g3 conformation based on experimental data. Additional stereospecific assignments of the methyl groups for ValB18 and LeuA23 were determined based on their NOE patterns. Hydrogen bonds were included in the structure calculations for all amide protons concluded to be slowly exchanging only after a suitable acceptor could be identified in the preliminary structures. In all cases, these hydrogen bonds were found between the backbone atoms within the elements of secondary structure. Three-dimensional structures were calculated using simulated annealing and energy minimization protocols from ARIA (23Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar) in the program CNS (24Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The protocol involves a high temperature phase with 4000 steps of 0.015 ps of torsion angle dynamics; a cooling phase with 4000 steps of 0.015 ps of torsion angle dynamics during which the temperature is lowered to 0 K; and finally, an energy minimization phase with 5000 steps of Powell minimization. The refinement in explicit water involves the following steps: heating to 500 K via steps of 100 K, each with 50 steps of 0.005 ps of Cartesian dynamics; 2500 steps of 0.005 ps of Cartesian dynamics at 500 K; and a cooling phase in which the temperature is lowered in steps of 100 K, each with 2500 steps of 0.005 ps of Cartesian dynamics. Finally, the structures were minimized with 2000 steps of Powell minimization. Protein structures were analyzed using PROMOTIF and PRO-CHECK and displayed using MOLMOL. Ramachandran analysis showed that 83% of the residues are in the most favored regions, with the remaining in the additionally allowed (15%) and generously allowed (2%). The coordinates representing the solution structure of H3 relaxin and the experimental restraints have been submitted to the Protein Data Bank (Code 2FHW). Peptide Synthesis—H3 relaxin was prepared as both its C-terminal acid and amide forms by solid-phase peptide synthesis of the separate A- and B-chains together with regioselective disulfide bond formation (25Lin F. Otvos Jr., L. Kumagai J. Tregear G.W. Bathgate R.A.D. Wade J.D. J. Pept. Sci. 2004; 10: 257-264Crossref PubMed Scopus (30) Google Scholar). Chemical characterization by reversed-phase HPLC and MALDI-TOF mass spectrometry confirmed the homogeneity of the products. Tryptic mapping followed by MALDI-TOF mass spectrometry showed that the disulfide bonds were in the correct insulin-like pairings. Biological Activity—H3 relaxin acid was tested for its ability to stimulate human LGR7, LGR8, and GPCR135 in parallel with the H3 relaxin amide peptide. As shown in Fig. 2, the activities of the peptides were identical. In LGR7-expressing cells, both peptides stimulated cAMP accumulation to levels similar to those achieved with H2 relaxin (H3 relaxin acid pEC50 = 8.08 ± 0.07 (n = 4) and H3 relaxin amide pEC50 = 8.19 ± 0.078 (n = 3)). In GPCR135-expressing cells, both peptides decreased forskolin-stimulated cAMP accumulation (H3 relaxin acid pEC50 = 9.30 ± 0.082 (n = 3) and H3 relaxin amide pEC50 = 9.25 ± 0.092 (n = 3)). Both peptides were unable to stimulate cAMP accumulation in LGR8-expressing cells at concentrations up to 1 μm (data not shown). NMR Diffusion Measurements—In light of the tendency of members of the insulin superfamily to aggregate, including H2 relaxin and insulin, it was important to establish whether any multimers of H3 relaxin were present under the conditions used for the NMR studies. This was assessed by measuring translational diffusion using pulsed-field gradient experiments with dioxane (hydrodynamic radius of 2.12 Å) as an internal standard. No significant difference in the hydrodynamic radius of H3 relaxin was observed between concentrations of 0.1 and 1 mm (14.2 and 14.4Å,respectively), indicating that there was no concentration-dependent aggregation. On the basis of the equation derived by Wilkins et al. (22Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (830) Google Scholar) (Rh = (4.75 ± 1.11)N0.29±0.02, where N is the number of residues in the protein and Rh is the hydrodynamic radius in Angstroms), the expected values for a H3 relaxin monomer (51 residues) and a H3 relaxin dimer (102 residues) would be ∼14.8 and ∼18.2 Å, respectively, confirming that H3 relaxin is monomeric under the conditions used for these studies. NMR Assignments and Structure Determination of H3 Relaxin—For the structural analysis of H3 relaxin, extensive two-dimensional NMR spectral data were recorded at 600 and 750 MHz on a sample containing ∼1 mm peptide. The spectral data were of high quality with excellent signal dispersion indicative of a well structured peptide. Resonance assignments were achieved using two-dimensional sequential assignment strategies, which, after analysis of spectra of both the H3 relaxin acid and amide forms at several temperatures, resulted in complete assignments for the peptide backbone and nearly complete assignment for side chain resonances. Interestingly, a number of residues showed broad lines; and in particular, significant line broadening was observed for a set of resonances, including SerA7, CysA10, CysA11, ArgA12, TrpA13, GlyA14, CysA15, CysB10, and PheB14, suggesting that conformational exchange is present in this region of the molecule. This exchange is present in this region of the molecule. This exchange is likely a result of a pro-R/pro-S disulfide bonds. In addition, several weak spin systems arising from a minor conformation were identified. These resonances were found correspond to the N-terminal region of the B-chain, i.e. AlaB2, AlaB3, ProB4, and TyrB5, and analysis of NOEs revealed that they were the result of a cis/trans-isomerization of ProB4. The trans-conformation is the major isomer (∼90%), with the cis-conformation corresponding to a minor conformation (∼10%), as evident from strong sequential Hαi-1-Hδi and Hαi-1-Hαi NOEs, respectively. The structural restraints derived from the NMR data and used for structure calculations were based exclusively on data recorded for the native acid form and included interproton distances, backbone and side chain dihedral angles derived from coupling constants, and restraints for hydrogen bonds deduced from amide exchange experiments. Structures were calculated by simulated annealing followed by refinement and energy minimization in explicit solvent (H2O). Distance restraints were derived from intensities of NOE cross-peaks in an NOE correlation spectrum recorded at 298 K with a mixing time of 100 ms. As expected for a 51-amino acid peptide, a number of cross-peaks were ambiguous at the first stage of the assignment due to degeneracy of chemical shifts, in particular in the methyl region. These ambiguities were resolved using an iterative approach in which preliminary structures were used to guide the assignments and by inclusion of ambiguous restraints. Hydrogen bonds were inferred from amide exchange behavior and introduced into the structure calculations once the preliminary structures indicated a suitable acceptor, which in all cases were found to be within elements of secondary structure. Description of the Three-dimensional Structure of H3 Relaxin—A summary of the NMR data, including sequential and medium-range NOEs, coupling constants, hydrogen exchange, and secondary Hα shifts, is presented in Fig. 3. These data provide a good indication of the presence of secondary structure, as both helical and extended conformations have typical "fingerprints." Helices are generally characterized by a large number of medium-range NOE contacts, small 3JHα-HN coupling constants, and negative secondary shifts, whereas β-sheets display large 3JHα-HN coupling constants and positive secondary shifts. The exchange rates between the backbone amide protons and the solvent can be measured by recording the decay in signal after dissolution of the peptide in D2O and provide information about the presence of hydrogen bonds, as amide protons involved in hydrogen bonds are protected from the solvent and display a slow exchange behavior. Although most amides in H3 relaxin exchange in minutes, a number are still visible >2 h after dissolution in D2O, and these could in all cases be correlated to hydrogen bonds in the structure. Fig. 4 shows a superposition of a family of 20 low energy structures representing the solution structure of H3 relaxin. Overall, the structure is well defined, with the exception of the N terminus of the B-chain, which is disordered and likely flexible in solution. The energetic and structural statistics for the structural family are summarized in Table 1. All structures have good energies and covalent geometry as evident from small deviations from ideal bond lengths and angles and the Ramachandran analysis, which showed that 83% of all non-Gly/Pro residues are in the most favored regions and that the remaining residues, all of which are in the more flexible regions of the peptide, are in the additionally allowed regions. It is clear that H3 relaxin adopts a typical relaxin/insulin fold, with the A-chain containing two α-helices (residues A1-A13 and A17-A24) separated by a short β-strand (residues A15-A17) and the B-chain containing a second β-strand (residues B5-B7) and an α-helix (residues B12-B22). The A-chain helices lie parallel to each other, forming a U-shaped arrangement. The B-chain helix is placed across the face of the U, roughly perpendicular to the axes of the A-chain helices. Enclosed between them is a significant hydrophobic core involving the side chains of, LeuA3, LeuA6, IleA20, LeuA23, CysA10, CysA15, LeuB9, CsyB10, PheB14, IleB15, ValB18, IleB19 and TrpB27. The observation that the Trp side chain interacts with the hydrophobic core as evident from a large number of NOEs to CysA24, CysB22, IleB15, ValB18, and IleB19 was surprising because, in the crystal structure of H2 relaxin, this region is disordered, and the Trp extends away from the core, being fully exposed to the solvent. The interaction appears to have become possible because of a shortening of the B-chain helix and a reversal of the peptide backbone due to turns formed by the B23-B26 (GGSR) segment.TABLE 1NMR and refinement statistics for protein structuresH3 relaxinNMR distance and dihedral constraintsDistance constraintsTotal inter-residue NOEs766Sequential (|i - j| = 1)311Medium-range (|i - j| < 4)213Long-range (|i - j| > 5)242Hydrogen bonds21Total dihedral angle restraintsΦ33ϰ116Structure statisticsViolations (mean ± S.D.)Distance constraints (>0.2 Å)0.4 ± 0.60Dihedral angle constraints (>2°)0Maximum distance constraint0.24violation (Å)Deviations from idealized geometryBond lengths (Å)0.00414 ± 0.00011Bond angles0.512 ± 0.017°Impropers0.412 ± 0.026°Average pairwise r.m.s.d.aPairwise root mean square deviation (r.m.s.d.) was calculated for 20 refined structures over residues A1-A24 and B3-B27. (Å)Heavy1.11Backbone0.50a Pairwise root mean square deviation (r.m.s.d.) was calculated for 20 refined structures over residues A1-A24 and B3-B27. Open table in a new tab Solution Structure of H3 Relaxin—In this study, we have determined the three-dimensional solution structure of H3 relaxin. H3 relaxin is well behaved in solution and adopts an insulin-like fold that is braced by the three disulfide bonds conserved throughout the family and that is characterized by three helical segments and a short double-stranded β-sheet. Although the structure is well defined overall, there is evidence for dynamic processes in several regions of the peptide. Most pronounced is the disorder at the N terminus of the B-chain, which is likely due to flexibility in this region. Supporting this suggestion is the observation of cis/trans-isomerization for ProB4, which results in two sets of resonances for the amino acids in this region. A similar isomerization has been observed in insulin (26Higgins K.A. Craik D.J. Hall J.G. Andrews P.R. Drug Des. Delivery. 1988; 3: 159-170Google Scholar), but there are no data suggesting that this feature is implicated in the biological function. More interesting is the observation of specific broadening of peaks from residues near the CysA10 -CysA15 and CysA11 -CysB10 disulfide bonds, with the extreme example
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