Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease.

The same heterozygous T -> C transition at nt 8567 of the von Willebrand factor (vWF) transcript was found in two unrelated patients with type III) von Willebrand disease, with no other apparent abnormality. In one family, both alleles were normal in the parents and one sister; thus, the mutation originated de novo in the proposita. The second patient also had asymptomatic parents who, however, were not available for study. The structural consequences of the identified mutation, resulting in the CyS2010 -> Arg substitution, were evaluated by expression of the vWF carboxyl-terminal domain containing residues 1366-2050. Insect cells infected with recombinant baculovirus expressing normal vWF sequence secreted a disulfide linked dimeric molecule with an apparent molecular mass of 150 kDa before reduction, yielding a single band of 80 kDa after disulfide bond reduction. In contrast, cells expressing the mutant fragment secreted a monomeric molecule of apparent molecular mass of 80 kDa, which remained unchanged after reduction. We conclude that CyS2010 is essential for normal dimerization of vWF subunits through disulfide bonding of carboxyl-terminal domains and that a heterozygous mutation in the corresponding codon is responsible for defective multimer formation in type III) von Willebrand disease.

Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport.

A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine. Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers. In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct. This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays. However, these experiments do not directly distinguish between a "one-gate" and a "two-gate" mechanism for the enzyme.

Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Identification and characterization of periplasmic proteins implicated in the adaptation of Helicobacter pylori to the human gastric niche

Helicobacter pylori è un batterio Gram-negativo in grado di colonizzare la mucosa gastrica umana e persistere per l'intero arco della vita dell'ospite. E' associato a patologie gastrointestinali, quali gastrite cronica, ulcere gastriche e duodenali, adenocarcinomi e linfomi gastrici. Si tratta di uno dei patogeni più diffusi, presente in circa metà della popolazione mondiale, e il solo che si è adattato a vivere nell'ambiente ostile dello stomaco umano. Molteplici sono i fattori di virulenza che permettono al batterio la colonizzazione della nicchia gastrica e contribuiscono, anche attraverso l' induzione di una risposta infiammatoria, a profonde modificazioni dell' omeostasi gastrica. Queste ultime si associano, ad esempio, all'iperproduzione di fattori proinfiammatori, ad alterazioni sia della regolazione della secrezione acida gastrica sia del ciclo cellulare e della morte cellulare programmata (apoptosi) delle cellule epiteliali gastriche, a disordini nel metabolismo del ferro e a carenze di elementi essenziali. Studi sulla diversità genetica di H. pylori osservata in ceppi isolati da varie regioni del mondo, dimostrano che tale batterio ha avuto una coevoluzione col genere umano attraverso la storia, ed è verosimile che H. pylori sia stato un costituente del microbiota gastrico per almeno 50.000 anni. Scopo della tesi è stato quello di identificare e caratterizzare proteine importanti per la colonizzazione e l'adattamento di H. pylori alla nicchia gastrica. In particolare gli sforzi si sono concentrati su due proteine periplasmatiche, la prima coinvolta nella difesa antiossidante (l'enzima catalasi-like, HP0485), e la seconda nel trasporto di nutrienti presenti nell'ambiente dello stomaco all'interno della cellula (la componente solubile di un ABC transporter, HP0298). La strategia utilizzata prevede un'analisi bioinformatica preliminare, l'ottenimento del gene per amplificazione, mediante PCR, dal genoma dell'organismo, la costruzione di un vettore per il clonaggio, l'espressione eterologa in E. coli e la successiva purificazione. La proteina così ottenuta viene caratterizzata mediante diverse tecniche, quali spettroscopia UV, dicroismo circolare, gel filtrazione analitica, spettrometria di massa. Il capitolo 1 contiene un'introduzione generale sul batterio, il capitolo 2 e il capitolo 3 descrivono gli studi relativi alle due proteine e sono entrambi suddivisi in un abstract iniziale, un'introduzione, la presentazione dei risultati, la discussione di questi ultimi, i materiali e i metodi utilizzati. La catalasi-like (HP0485) è una proteina periplasmatica con struttura monomerica, appartenente ad una famiglia di enzimi a funzione per la maggior parte sconosciuta, ma evolutivamente correlati alla ben nota catalasi, attore fondamentale nella difesa di H. pylori, grazie alla sua azione specifica di rimozione dell'acqua ossigenata. HP0485, pur conservando il fold catalasico e il legame al cofattore eme, non può compiere la reazione di dismutazione dell'acqua ossigenata; possiede invece un'attività perossidasica ad ampio spettro, essendo in grado di accoppiare la riduzione del perossido di idrogeno all'ossidazione di diversi substrati. Come la catalasi, lavora ad alte concentrazioni di aqua ossigenata e non arriva a saturazione a concentrazioni molto elevate di questo substrato (200 mM); la velocità di reazione catalizzata rimane lineare anche a questi valori, aspetto che la differenzia dalle perossidasi che vengono in genere inattivate da concentrazioni di perossido di idrogeno superiori a 10-50 mM. Queste caratteristiche di versatilità e robustezza suggeriscono che la catalasi-like abbia un ruolo di scavenger dell'acqua ossigenata e probabilmente anche un'altra funzione connessa al suo secondo substrato, ossia l'ossidazione di composti nello spazio periplasmatico cellulare. Oltre alla caratterizzazione dell'attività è descritta anche la presenza di un ponte disolfuro, conservato nelle catalasi-like periplasmatiche, con un ruolo nell'assemblaggio dell'eme per ottenere un enzima attivo e funzionale. La proteina periplasmatica HP0298, componente di un sistema di trasporto ABC, è classificata come trasportatore di dipeptidi e appartiene a una famiglia di proteine in grado di legare diversi substrati, tra cui di- e oligopeptidi, nichel, eme, glutatione. Benchè tutte associate a trasportatori di membrana batterici, queste proteine presentano un dominio di legame al substrato che risulta essere conservato nei domini extracellulari di recettori specifici di mammifero e uomo. Un esempio sono i recettori ionotropici e metabotropici del sistema nervoso. Per caratterizzare questa proteina è stato messo a punto un protocollo di ligand-fishing accoppiato alla spettrometria di massa. La proteina purificata, avente un tag di istidine, è stata incubata con un estratto cellulare di H. pylori per poter interagire con il suo substrato specifico all'interno dell'ambiente naturale in cui avviene il legame. Il complesso proteina-ligando è stato poi purificato per cromatografia di affinità e analizzato mediante HPLC-MS. L'identificazione dei picchi differenziali tra campioni con la proteina e 5 campioni di controllo ha portato alla caratterizzazione di pentapeptidi particolarmente ricchi in aminoacidi idrofobici e con almeno un residuo carico negativamente. Considerando che H. pylori necessita di alcuni aminoacidi essenziali, per la maggior parte idrofobici, e che lo stomaco umano è particolarmente ricco di peptidi prodotti dalla digestione delle proteine introdotte con il cibo, il ruolo fisiologico di HP0298 potrebbe essere l'internalizzazione di peptidi, con caratteristiche specifiche di lunghezza e composizione, che sono naturalmente presenti nella nicchia gastrica.

Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[ 6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI1[6,5] is similar to9:1 regardless of whether trypsin is incubated with SFTI-1[ 6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[ 6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[ 6,5] revealed high heterogeneity, and multiple species of SFTI-1[ 6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.

Twists, knots, and rings in proteins: Structural definition of the cyclotide framework

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Mobius cyclotides) and knots (a knotted arrangement of the disulfide bonds).

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

The chemistry and biology of human relaxin-3

A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.