GPCR homology model template selection benchmarking: Global versus local similarity measures

Abstract

G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for drug development. GPCR ligand interaction studies often have a starting point with either crystal structures or comparative models. The majority of GPCR do not have experimentally-characterized 3-dimensional structures, so comparative modeling, also called homology modeling, is a good structure-based starting point. Comparative modeling is a widely used method for generating models of proteins with unknown structures by analogy to crystallized proteins that are expected to exhibit structural conservation. Traditionally, comparative modeling template selection is based on global sequence identity and shared function. However high sequence identity localized to the ligand binding pocket may produce better models to examine protein-ligand interactions. This in silico benchmark study examined the performance of a global versus local similarity measure applied to comparative modeling template selection for 6 previously crystallized, class A GCPR (CXCR4, FFAR1, NOP, P2Y12, OPRK, and M1) with the long-term goal of optimizing GPCR ligand identification efforts. Comparative models were generated from templates selected using both global and local similarity measures. Similarity to reference crystal structures was reflected in RMSD values between atom positions throughout the structure or localized to the ligand binding pocket. Overall, models deviated from the reference crystal structure to a similar degree regardless of whether the template was selected using a global or local similarity measure. Ligand docking simulations were performed to assess relative performance in predicting protein-ligand complex structures and interaction networks. Calculated RMSD values between ligand poses from docking simulations and crystal structures indicate that models based on locally selected templates give docked poses that better mimic crystallographic ligand positions than those based on globally-selected templates in five of the six benchmark cases. However, protein model refinement strategies in advance of ligand docking applications are clearly essential as the average RMSD between crystallographic poses and poses docked into local template models was 9.7 Å and typically less than half of the ligand interaction sites are shared between the docked and crystallographic poses. These data support the utilization of local similarity measures to guide template selection in protocols using comparative models to investigate ligand-receptor interactions.

Keywords: Comparative modeling; Comparative protein modeling; Deorphanization; G protein-coupled receptor; GPCR; Homology modeling; Ligand identification; Template selection.

Figure 1.

Superposition of 50 representative GPCR structures. Fusion partners were deleted prior to superposition. Backbone structures in the TM region show strong structural conservation. TM segments 1 and 4 are labeled, TM segments 5–7 are behind TM segments 1–4 in this view. Specific GPCR structures used are shown in Table S1.

Figure 2:

Names and structures of ligands present in reference crystal structures and cross-docking target crystal structures. Ligands present in reference crystal structures were used in docking studies and are shown in the ionization state used in docking studies. The first line of text under each ligand indicates the ligand abbreviation used in the PDB, followed by the receptor name and the four-character PDB entry code.

Figure 3:

Panel A) Reference crystal structure of P2Y12 (red ribbons) with the 53 unique of 61 residues for the PRMSD61 shown in grey and the 8 residues common to the PRMSD61 and PRMSD8 shown in teal. Panel B) Superposition of P2Y12 reference (red ribbons and alternate crystal structures (teal and pink ribbons for 4NTJ and 4PXZ, respectively), local and global template models from structure-independent alignment (grey and blue ribbons, respectively), local and global template model from structure-dependent alignment (yellow and orange ribbons, respectively).

Figure 4:

Superposition of docked and crystallographic poses for three examples: OPRK (Panels A and D), FFAR1 (Panels B and E), and CXCR4 (Panels C and F). Reference crystal structures are shown in red ribbons. Induced fit causes slight sidechain movements. Yellow ribbons resulted from induced fit docking. A-C) View perpendicular to helical axes. TM 6 and TM 7 are hidden to allow viewing into the binding pocket. D-F) View from extracellular space.

Figure 5.

Superposition of docked and crystallographic poses for JDC in OPRK. OPRK reference (4DJH) and cross-docking target crystal structures (6B73) are shown with red and teal ribbons. OPRK local and global template homology models from structure-independent alignments are shown with grey and blue ribbons. OPRK local and global template homology models from structure-based alignments are shown with yellow and orange ribbons. Ligand colors are matched to the ribbon color of the docking target. TM 6 and 7 are hidden to allow view into binding pocket. A,B,C, E, F: View perpendicular to helical axes. D: View from extracellular space.