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 25-32 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.

Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region

“…The potential role of the NTF, how it may be positioned compared to the CTF and how it may affect its structure is not known so far. Modeling the highly flexible loops remains challenging, although some studies already focused on modeling of GPCR ECLs (Wink et al, 2019). In particular, ECL2 is the largest ECL in GPR116, as in many other GPCRs, and the disulfide bridge with TM3 does not introduce sufficient constraints to develop a better defined model.…”

Photocleavable proteins that undergo fast and efficient dissociation

“…The circular cp linker (residues 147–172), which was not resolved in the crystal structure of the photo-converted PhoCl1, was modelled in Rosetta (version 2018.33.60351) using the kinematic closure (KIC) with fragment protocol. 57 Briefly, 10 000 models were generated and the structure with the largest negative score was selected to represent the starting conformation of the cp linker. The experimental structure of the PhoCl1 red state structure, edited to include the parameterized RCP chromophore and the cp linker, was subsequently solvated in LEaP (AmberTools19) with explicit water (TIP3P) and 1 sodium counterion in an octagon periodic boundary condition with each side no closer than 30 Å away from the protein.…”

“…56). The circular cp linker (residues 147-172), which was not resolved in the crystal structure of the photo-converted PhoCl1, was modelled in Rosetta (version 2018.33.60351) using the kinematic closure (KIC) with fragment protocol 57 .…”

Linearised loop kinematics to study pathways between conformations

“…However, while these algorithms have been game-changing, e.g. for loop modelling with fragments [27,25,48], they are not well suited to study conformational changes. That is because they do no permit close control of the sampling direction.…”