Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Abstract

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 (Cys27-Cys82, Cys40-Cys72, and Cys57-Cys104) was determined by site-directed mutagenesis. The secondary structure (alpha-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 A, 4.1 A, and 4.0 A, respectively. The model of RAMP1 suggested that Phe93, Tyr100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer.

FIGURE 1

Sequence alignments of human RAMP1, RAMP2, and RAMP3. Signal peptide shown in italics and underlined; domain implicated in adrenomedullin binding shown underlined; and W, Trp⁷⁴ required for high affinity BIBN4096BS binding. N, glycosylated residue. Conserved cysteines shaded gray. Residues labeled with a single dot are conserved in the extracellular domains of RAMP1 and RAMP3; residues labeled with two dots are conserved in the extracellular domains of all RAMPs.

FIGURE 2

Primary sequence of extracellular residues (26–109) of hRAMP1. Shown on the diagram are the consensus α-helical regions (dotted) as predicted by JPED and SAMT02 servers and the final positions of the helices (solid) after MD refinement. Also shown on the diagram is the disulphide pattern Cys²⁷⁽¹⁾-Cys82(5), Cys⁴⁰⁽²⁾-Cys⁷²⁽⁴⁾ and Cys⁵⁷⁽³⁾-Cys¹⁰⁴⁽⁶⁾ as suggested by in silico prediction and confirmed by mutagenesis.

FIGURE 3

CGRP-stimulated cAMP production in mutated RAMPs. Cos 7 cells were transfected with CL/WT hRAMP1 or CL/mutant hRAMP1 and assayed for CGRP-stimulated cAMP production. (▪) WT type receptors. Mutant receptors are as indicated. Data are representative of three similar experiments. Points are the mean ± SE of triplicate determinations.

FIGURE 4

Comparison of the refined ab initio models (left) and the corresponding x-ray crystal structure (right) for PDB:1HP8, PDB:1V54 and PDB:1T0P after 50ns of MD simulation. The structures were compared and revealed a C_α RMSD of 3.8 Å, 4.1 Å, and 4.0 Å for the PDB:1HP8, PDB:1V54, and PDB:1T0P x-ray crystal structures, respectively.

FIGURE 5

Ab initio model of hRAMP1. Panels A and B show the architecture of hRAMP1 with three helices and interconnecting loops. (A) Previous studies have highlighted Phe⁹³ and Tyr¹⁰⁰ as being important in cell surface expression. The ab initio model of hRAMP1 reveals that these residues are located in a cleft between helix1 and helix3. (B) Location of Phe^92, which has been shown to affect ligand binding, but has little effect on cell surface expression. Also shown in B is the location of Trp⁷⁴, a residue that has been implicated in the high affinity binding of the non-peptide antagonist BIBN4096BS.