A benchmark study of loop modeling methods applied to G protein-coupled receptors

Abstract

G protein-coupled receptors (GPCR) are important drug discovery targets. Despite progress, many GPCR structures have not yet been solved. For these targets, comparative modeling is used in virtual ligand screening to prioritize experimental efforts. However, the structure of extracellular loop 2 (ECL2) is often poorly predicted. This is significant due to involvement of ECL2 in ligand binding for many Class A GPCR. Here we examine the performance of loop modeling protocols available in the Rosetta (cyclic coordinate descent [CCD], KIC with fragments [KICF] and next generation KIC [NGK]) and Molecular Operating Environment (MOE) software suites (de novo search). ECL2 from GPCR crystal structures served as the structure prediction targets and were divided into four sets depending on loop length. Results suggest that KICF and NGK sampled and scored more loop models with sub-angstrom and near-atomic accuracy than CCD or de novo search for loops of 24 or fewer residues. None of the methods were able to sample loop conformations with near-atomic accuracy for the longest targets ranging from 25 to 32 residues based on 1000 models generated. For these long loop targets, increased conformational sampling is necessary. The strongly conserved disulfide bond between Cys3.25 and Cys45.50 in ECL2 proved an effective filter. Setting an upper limit of 5.1 Å on the S-S distance improved the lowest RMSD model included in the top 10 scored structures in Groups 1-4 on average between 0.33 and 1.27 Å. Disulfide bond formation and geometry optimization of ECL2 provided an additional incremental benefit in structure quality.

Keywords: Comparative modeling; GPCR; Homology modeling; Loop modeling.

Fig. 1. Extracellular loop comparison of seventeen superposed GPCR crystal structures

The PDB IDs of the superposed GPCR crystal structures above are as follows: Bovine RHO (1GZM, green), B2AR (2RH1, orange), B1AR(2VT4, magenta), Squid RHO (2Z73, gold), AA2AR (3EML, cyan), DRD3 (3PBL, pink), H1R (3RZE, maroon), ACM2 (3UON, light blue), CXCR4 (3ODU, brown), S1PR1 (3V2Y, dark green), PAR1 (3VW7, yellow), NTR1 (4GRV, blue), ACM3 (4DAJ, light brown), OPRK (4DJH, purple), OPRM (4DKL, dark blue), NOP (4EA3, salmon), OPRD (4EJ4, dark brown).

Fig. 2. ECL2 comparison of GPCR that share high percentage sequence identity

The DRD3 (3PBL, orange), DRD2 (6CM4, cyan), and 5-HT2C (6BQH, magenta) crystal structures are shown to highlight the structurally variable ECL2 segments.

Fig. 3. Overview schematic of Rosetta loop modeling process

Fig. 4. Overview schematic of MOE loop modeling process

Fig. 5. Energy function comparison with Rosetta NGK loop modeling

Comparisons of the energy function influence on loop modeling performance is shown for the shortest GPCR ECL2 targets in the benchmark. The lowest RMSD models (LRM), top scored models (T1), lowest RMSD models in the top 10 scored (T10), and lowest RMSD models in the top 25 scored (T25) are shown from 1000 models generated for each ECL2 target using NGK loop modeling. The number in parentheses represents the ECL2 length.

Fig. 6. Group 1 Loop Modeling Benchmark set

(a) The RMSD values of the LRM and T1 are shown out of 1000 models generated for Group 1 ECL2 targets using NGK, KICF, CCD, and MOE loop modeling algorithms. In total, the LRM had sub-atomic accuracy in two and three cases when using the NGK and KICF algorithms, respectively. Additionally, the LRM had near-atomic accuracy in seven cases when using the KICF algorithm and six cases when using the NGK or CCD algorithms. (b) The RMSD values of the T1 is compared to the T10 and T25 using NGK, KICF, CCD, and MOE.

Fig. 7. ECL2 models of ACM4 superposed with reference structure

(a) The LRM (orange) and T1 (magenta) out of 1000 total models generated using NGK loop modeling had RMSD values of 0.34 Å and 0.40 Å to the reference structure (green). (b) The LRM and T1 out of 1000 total models generated using KICF loop modeling had RMSD values of 0.35 Å and 0.62 Å to the reference structure.

Fig. 8. ECL2 models of CXCR4 superposed with reference structure

(a) The LRM (orange) and T1 (magenta) out of 1000 total models generated using NGK loop modeling had RMSD values of 3.08 Å and 7.02 Å to the reference structure (green). (b) The LRM and T1 out of 1000 total models generated using KICF loop modeling had RMSD values of 0.50 Å and 4.19 Å to the reference structure.

Fig. 9. Group 2 Loop Modeling Benchmark Set

(a) The RMSD values of the LRM and T1 are shown out of 1000 models generated for Group 2 ECL2 targets using NGK, KICF, and CCD algorithms. In total, the LRM had sub-atomic accuracy in one and two cases when using the NGK and KICF algorithms, respectively. Additionally, the LRM had near-atomic accuracy in four cases when using the KICF algorithm and in three cases when using the NGK or CCD algorithms. (b) The RMSD values of the T1 is compared to the T10 and T25 using NGK, KICF, and CCD. The upper and lower dotted lines represent the near-atomic and sub-angstrom accuracy thresholds, respectively.

Fig. 10. ECL2 models of CB1 superposed with reference structure

(a) The LRM (orange) and T1 (magenta) out of 1000 models total using NGK loop modeling had RMSD values of 0.85 Å and 4.32 Å to the reference structure (green). (b) The LRM and T1 when KICF loop modeling was used had RMSD values of 0.82 Å and 1.93 Å to the reference structure.

Fig. 11. ECL2 models of SMO superposed with reference structure

(a) Side view of SMO crystal structure (PDB:4JKV) highlighting the native ECL2 (green) buried underneath ECL1 (gray) and the ECD linker (salmon). (b) Top view of the extracellular side of the SMO crystal structure (c) The LRM (orange), T1 (magenta), and T10 (cyan) using the NGK algorithm had RMSD values of 6.22 Å, 17.0 Å, and 12.7 Å to the reference structure, respectively. (d) The LRM, T1, and T10 using the KICF algorithm had RMSD values of 1.58 Å, 19.9 Å, and 6.89 Å to the reference structure, respectively. The ECD linker and ECL1 regions were hidden in panels C and D to visualize ECL2 and models clearly.

Fig. 11. ECL2 models of SMO superposed with reference structure

(a) Side view of SMO crystal structure (PDB:4JKV) highlighting the native ECL2 (green) buried underneath ECL1 (gray) and the ECD linker (salmon). (b) Top view of the extracellular side of the SMO crystal structure (c) The LRM (orange), T1 (magenta), and T10 (cyan) using the NGK algorithm had RMSD values of 6.22 Å, 17.0 Å, and 12.7 Å to the reference structure, respectively. (d) The LRM, T1, and T10 using the KICF algorithm had RMSD values of 1.58 Å, 19.9 Å, and 6.89 Å to the reference structure, respectively. The ECD linker and ECL1 regions were hidden in panels C and D to visualize ECL2 and models clearly.

Fig. 12. Group 3 Loop Modeling Benchmark Set

(a) The RMSD values of the LRM and T1 are shown out of 1000 models generated for Group 3 ECL2 targets using the NGK, KICF, and CCD algorithms. In total, the LRM had sub-atomic accuracy in two cases when using the KICF algorithm. Additionally, the LRM had near-atomic accuracy in three cases when using the KICF algorithm and in single cases when using the NGK or CCD algorithms. (b) The RMSD values of the T1 is compared to the T10 and T25 using NGK, KICF, and CCD. The upper and lower dotted lines represent the near-atomic and sub-angstrom accuracy thresholds, respectively.

Fig. 13. ECL2 models of P2YR1 superposed with reference structure

(a) The LRM (orange), T1 (magenta), and T10 (cyan) out of 1000 models generated using NGK loop modeling had RMSD values of 2.44 Å, 6.53 Å, and 2.55 Å to the reference structure (green), respectively. (b) In this case, the LRM was also the T1 (magenta) when KICF loop modeling was used (LRM = T1 = 0.63 Å RMSD to reference structure).

Fig. 14. ECL2 models of B2AR superposed with reference structure

(a) The LRM (orange), T1 (magenta), and T10 (cyan) out of 1000 models generated using NGK loop modeling had RMSD values of 3.84 Å, 18.1 Å, and 6.81 Å to the reference structure (green), respectively. (b) The LRM, T1, and T10 generated using KICF loop modeling had RMSD values of 2.60 Å, 18.6 Å, and 5.04 Å to the reference structure, respectively.

Fig. 15. Group 4 Loop Modeling Benchmark Set

(a) The RMSD values of the LRM and T1 are shown out of 1000 models generated for Group 4 ECL2 targets using the NGK, KICF, and CCD algorithms. The T10 and T25 are shown out of 1000 models generated for Group 4 ECL2 targets using NGK, KICF, and CCD. (b) The RMSD values of the T1 is compared to the T10 and T25 using NGK, KICF, and CCD. The upper and lower dotted lines represent the near-atomic and sub-angstrom accuracy thresholds, respectively.

Figure 16.. PAR2 ECL2 Models (KICF) and Reference Structure.

(a) Top ten scored PAR2 ECL2 models produced within 1000 structures generated using KICF in Rosetta. (b) PAR2 crystallographic reference structure. (c) Top ten scored PAR2 ECL2 models after filtering 1000 structures generated using KICF in Rosetta for Cys3.25-Cys45.50 S-S distances 5.1 ≤ Å.

Figure 17.. AA2AR ECL2 Models (KICF) and Reference Structure.

(a) Top ten scored AA2AR ECL2 models produced within 1000 structures generated using KICF in Rosetta. (b) AA2AR crystallographic reference structure. (c) Top ten scored AA2AR ECL2 models after filtering 1000 structures generated using KICF in Rosetta for Cys3.25-Cys45.50 S-S distances 5.1 ≤ Å.