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^sup 27^-Cys^sup 82^, Cys^sup 40^-Cys^sup 72^, and Cys^sup 57^-Cys^sup 104^) was determined by site-directed mutagenesis. The secondary structure (α-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:1 V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 [Angstrom], 4.1 [Angstrom], and 4.0 [Angstrom], respectively. The model of RAMP1 suggested that Phe^sup 93^, Tyr^sup 100^, and Phe^sup 101^ form a binding interface for CLR, whereas Trp^sup 74^ and Phe^sup 92^ may interact with ligands that bind to the CLR/RAMP1 heterodimer.

G-protein-coupled receptors (GPCRs) represent one of the largest protein families within the human genome. They have a characteristic architecture, consisting of seven transmembrane (TM) helices. Ligands hind to the extracellular face of the receptor or to a pocket formed within the TM region. In contrast, G-proteins hind to the intracellular face of the receptor.

Until recently, GPCRs were considered to act essentially as monomers. However, there is now considerable evidence that many form dimers or other oligomers (1). Most attention has been focused on dimers between GPCRs, but other proteins can also be involved. These include the family of receptor activity modifying proteins (RAMPs). These were first identified as partners for the calcitonin receptor-like receptor (CLR). CLR by itself is unable to bind any ligand; however, in the presence of RAMP1 it functions as a receptor for calcitonin gene-related peptide (CGRP). whereas in the presence of RAMP2 it becomes an adrenomedullin receptor. The CLR/RAMP3 complex also preferentially binds AM, but it has a greater affinity for CGRP than CLR/ RAMP2 (2). Subsequently it has been shown that RAMPs can associate with a number of other receptors, including the calcitonin, parathyroid hormone 1 and 2, vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPAC^sub 1^, VPAC^sub 2^), glucagons, and calcium-sensing GPCRs (3-5).

All three RAMPs are thought to he built around a common architecture (2,6) (Fig. 1). They have a short, intracellular C-terminus followed by a single TM region. The largest pail of the protein is the extracellular domain: ~90 amino acids for RAMP1 and RAMP3, whereas for human RAMP2 this domain is 13 residues longer. All RAMPs have four conserved cysteine residues; RAMP1 and RAMP3 have an additional pair.

It seems that the N-terminus is the major determinant of ligand binding (7.8). The structure-function relationship for RAMP2 and RAMP3 have been investigated by use of protein chimeras; these have identified residues 86-92 of human RAMP2 and 59-65 of human RAMP3 as key epitopes for AM binding (9). Deletion analysis of human RAMP3 suggested that residues 91-103 formed an important epitope for CGRP binding (10). In human RAMP1, Trp^sup 74^ is important for high-affinity binding of BIBN4096BS, a nonpeptide antagonist of CGRP; the mutation W74K substantially reduced antagonist affinity (11). There is no structural explanation for the effect of any of these mutants, and it is unclear whether the residues or epitopes make direct contact with the ligands or act indirectly to stabilize ligand binding sites. In addition, the cysteines in the N-terminus probably form disulfide bonds. Although some information has been obtained from previous studies (12), to date, there has not been any systematic mutagenesis study of their topology.

In this study we have produced mutant RAMP1 constructs which incorporate all possible pairwise combinations of Cys to Ala mutants, to determine the organization of the disullide bond network in hRAMP1. In addition, we have produced an ab initio molecular model of RAMP1 which is entirely consistent with the mutagenesis data presented in this study and also provides a mechanistic basis for mutagenesis data previously published by other laboratories.