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.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

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 Trp(B27) 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.

The absolute structural requirement for a proline in the P3 '-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp(14) was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro(13) and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.

Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

The pharmacology of adrenomedullin receptors and their relationship to CGRP receptors

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM 22-52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP8-37 is either equipotent or more effective as an antagonist than AM22-52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP 1 and AM2 receptors and AM could activate both AM 1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2. Copyright © 2004 Humana Press Inc. All rights of any nature whatsoever reserved.