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David Space

Posted on February 24, 2016


Date - February 24, 2016
1:00 pm


Ph.D. Thesis Defense – 752
Title: “Construction of a Homology Model of the Orexin-1 Receptor in its Active and Inactive States and Subsequent Molecular Dynamics Experiments in a Simulated Lipid Bilayer”


The purpose of Phase I of this research was to construct two homology models, one each inactive (R state) and active (R* state), of the orexin-1 receptor (ox1r).  The ox1r is a Class A (rhodopsin-like) G protein-coupled receptor (GPCR) that couples to Gq protein. Homology models were built using an existing crystal structure of a Class A GPCR.  The adenosine A2A receptor, beta-2 adrenergic receptor (β2-AR), and rhodopsin were early candidates for the template, and the β2-AR was chosen.

The helices were created by using Conformational Memories to select helix conformations that fit the β2-AR template, and the adenosine A2A receptor’s helix 7-8 assembly was used as well.  Modeller was used to construct the loops and termini, and the receptor models were palmitoylated.  The ligands 1-(5-(2-Fluoro-phenyl)-2-methylthiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042), and the orexin-A peptide [pyroglutamate]PLPDCCRQKTCSCRLYELLHGAGNHAAGILTL-NH2 with residues C6-C12 and C7-C14 disulfide-bonded, were docked in the ox1r R and R* states, respectively.  SB-674042 was docked so that it can hold the ox1r R state’s Y6.48 in g+ and that its amide can accept a hydrogen bond from the ox1r R state’s Q3.32.  Orexin-A was docked so that it can hold the ox1r R* state’s Y6.48 in trans and that orexin-A’s R8 donates a hydrogen bond to the ox1r R* state’s H7.39, which in turn donates a hydrogen bond to orexin-A’s [pyroglutamate]1 backbone amide oxygen.  Modeller was used to construct the loops and termini, and the receptors were palmitoylated.  With the release of the orexin-2 receptor (ox2r) crystal structure, helices created by Conformational Memories were selected based on their fit to the ox2r, with the ox2r helix 7-8 assembly used accordingly.  The ligands were again docked in their respective receptors to create the same principal interactions as before.  Loops and termini were again created with Modeller, and the receptors were palmitoylated.

The purpose of Phase II was to examine the homology models’ behavior in a simulated lipid bilayer environment.  To do so, CHARMM force field parameters were created based on the orexin-1 receptor antagonist SB-674042 and on the protein residue pyroglutamate.  The result was a list of new parameters that were suitable for use in the Molecular Dynamics simulation, and in turn, the results of the lipid bilayer simulation of each ligand/receptor complex indicated that each complex was stable and retained important GPCR features.