Structure modeling of all identified G protein-coupled receptors in the human genome

Abstract

G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness of the in silico models for GPCR functional analysis. All predicted GPCR models are freely available for noncommercial users on our Web site (http://www.bioinformatics.buffalo.edu/GPCR).

Figure 1. Initial Templates from PROSPECTOR_3 and the Final TASSER Model of Highest Cluster Density Superposed on the Bovine RH Crystal Structure

Blue to red runs from N- to C-terminus. The numbers are the RMSD to native. Images are from RASMOL [120].

Figure 2. Application of TASSER to Membrane Proteins

TASSER was applied to a benchmark set of 38 membrane proteins with structures in the PDB. RMSD to native for final models of TASSER versus RMSD to native for initial templates from PROSPECTOR_3. All points beneath the 45° line indicate an improvement in the TASSER model over the initial template. All template alignments with a sequence identity greater than 30% were excluded from consideration.

Figure 3. C-Score Distribution of the Predicted Models for the 907 GPCR Sequences

The C-score histogram for the PDB benchmark proteins [20] is shown for comparison, where dark gray denotes those models with an RMSD less than 6.5 Å to native and the light gray those models whose RMSD greater than 6.5 Å. The C-score is defined as in Equation 1. Inset: The cumulative foldable fraction calculated under the assumption that the GPCR proteins have the same correlation between success and C-score as that of the PDB benchmark proteins.

Figure 4. Conformational Changes of the Predicted TASSER Models from the Crystal Structure of Bovine RH

Data are the average from those targets where bovine RH is a template with C-score greater than 1.3 (red diamonds). The green triangles denote the TASSER model for bovine RH when bovine RH itself is used as the template (ten missed residues in 1f88 are inserted in the TASSER modeling). This shows the inherent resolution of the TASSER model. (A) Average distance of each residue of the TASSER models from the bovine RH template along the sequence. TM helices are marked in gray. (B) Percentage of all helices with helix angle changes below the indicated thresholds.

Figure 5. Consistency of Mutagenesis Studies with TASSER Predictions

TASSER models for gonadotropin hormone-release receptor, adenosine 3 receptor, mu opioid receptor, and melatonin 2 Receptor are shown with experimental determined residue interactions highlighted as spheres (green, ligand binding; yellow, disulfide bond; red, residue-residue interaction).