This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.106.084582v1 91/2/662    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Simms, J. Articles by Poyner, D. R. Search for Related Content PubMed PubMed Citation Articles by Simms, J. Articles by Poyner, D. R.

Characterization of the Structure of RAMP1 by Mutagenesis and Molecular Modeling

John Simms ^*, Debbie L. Hay , Mark Wheatley  and David R. Poyner ^*

^* School of Life and Health Sciences, Aston University, Birmingham, United Kingdom; School of Biosciences, University of Auckland, Auckland, New Zealand; and School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Correspondence: Address reprint requests to David Poyner, School of Life and Health Sciences, Aston University, Birmingham, B4 7ET UK. Tel.: 44-(0)121-204-3997; Fax: 44 (0)121-359-5142; E-mail: d.r.poyner{at}aston.ac.uk.

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 (-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, Tyr¹⁰⁰, and Phe¹⁰¹ form a binding interface for CLR, whereas Trp⁷⁴ and Phe⁹² may interact with ligands that bind to the CLR/RAMP1 heterodimer.