Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

¹ Aston University
² Univeristy of Auckland
³ Univerisity of Birmingham

^* To whom correspondence should be addressed. E-mail: d.r.poyner{at}aston.ac.uk.

Submitted on March 7, 2006
Revised on March 29, 2006
Accepted on 30 March 2006

	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 (-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 (MD) 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 3.8Å, 4.1Å and 4.0Å 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.

Key Words: CGRP, ab-initio modeling, calcitonin receptor-like receptor, disulfide bond prediction, secondary structure prediction

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