Computational protocol: An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

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Protocol publication

[…] CTR and CLR full-length sequences were aligned using Tcoffee []. A multiple template design was used to generate the CTR ECD. CLR ECD, chain A from 3N7S, was used as a template structure for CTR ECD. The first loop domain of chain A (3N7S) is not elucidated. Therefore, the loop domain of 3N7S (chain B) was used as the template for this loop domain in the CTR ECD. Modeller9v8 [] was used to generate 500 models. The models were ranked by the Modeller9v8 energy objective function. The top 10 structures were retained and the stereochemical quality was assessed by PROCHECKv3.5.4. []. On the basis of overall and residue-by-residue geometry a structure was selected. The ProPka program [] via the PDBQPR server [] was used to assign the protonation states of the titratable groups in CTR ECD, using the CHARMM parameters set at pH 7.0. The CHARMM (c35b3) module Screened Coulomb Potentials Implicit Solvent Model was used to minimize the model. 100 steps of steepest descent were conducted followed by adopted basis Newton-Raphson minimization until convergence was met.To model CGRP and rat amylin bound to the AMY receptor, a similar approach was used to that for the modelling of the receptor without ligand, but the structure of a CGRP analogue bound to the CGRP receptor ECD was used as a template (PDB ID: 4RWG). This was mutated in-silico to give the structures of either human CGRP or amylin. Subsequent steps were as described above. PDB files are available as (ctr_ecd; ctr_r1; ctr-ramp1-cgrpmut; ctr-ramp1-ramy). [...] Homology models of the ECD of the CTR alone and the CTR ECD docked to the RAMP1 ECD were based on the CLR/RAMP1 ECD structure 3N7R using a Clustal Omega alignment. With Modeller v9.10, 1 000 models were generated and scored using OPUS_PSP. The best scoring model was visually inspected before minimization using the Amber99sb ILDN force-field as implemented in GROMACS. For MD simulations, the minimized protein was placed centrally in a charge neutral cubic box (x=10 nm) consisting of TIP3 water molecules, 150 mm NaCl and simulated for 1 μs. Principal component analysis of the resulting trajectories was used to identify the main eigenvectors and the root mean square fluctuation calculated. […]

Pipeline specifications

Software tools T-Coffee, PROPKA, Clustal Omega, MODELLER, OPUS-PSP, GROMACS
Application Protein structure analysis
Chemicals Calcitonin