On the application of the MARTINI coarse-grained model to immersion of a protein in a phospholipid bilayer

Abstract

An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions.

Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function

“…A similar procedure was used to generate CG models of the four proteins in a POPC bilayer in aqueous solution to that described in our previous work (26). The initial all-atom (AA) protein models were converted to MARTINI CG models using the martinize.py script (http://cgmartini.nl), and the TM-helix was immersed in a pre-equilibrated modeled CG POPC lipid bilayer consisting of 594 POPC molecules.…”

“…The simulation procedure was the same as described in our previous work (26). Each simulation started with a short steepest-descent energy minimization until the maximal force on a CG particle was less than 10 kJ/mol,nm.…”

“…The back conversion of the protein was done using the scripts backward.py and initram.sh, available at the MARTINI website (http://cgmartini.nl) (31). In previous studies (26,29), the globular domain residues of the crystal structure were superimposed on the back-mapped structure to preserve the side-chain interactions in the heme cofactor binding pocket. However, during the CG simulations of mtCYP17, the positions of the TM-helix and linker region changed such that they developed interactions with the F-G loop of the globular domain and influenced the conformation of the F-G loop and its interactions with the membrane.…”

“…The systems were simulated using NAMD 2.10 (35). The systems were first energy minimized with harmonic restraints, with a force constant gradually decreasing from 1000 to 0 kcal/mol,Å 2 on nonhydrogen protein and lipid atoms, as described in (26). Then equilibration was performed with a time step of 1 fs in a constant surface area, pressure, and semiisotropic ensemble for 1.5 ns with gradually decreasing harmonic restraints (from 100 to 0 kcal/mol,Å 2 ) on nonhydrogen protein and lipid atoms.…”

“…They are important for stabilization of the TM-helix in the bilayer and for protein sorting and retention of CYPs in the ER membrane (4). For example, it has been observed that retention of the TM-helix of CYP 2C1 in the ER membrane is dependent on the hydrophobicity of the TM domain (residues [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and on the hydrophilic residues (21-23 KQS) at the beginning of the linker region (residues [21][22][23][24][25][26][27][28]. Mutation of the linker, the hydrophobic TM domain, or both regions resulted in the expulsion of CYP 2C1 from the ER membrane and affected direct retrieval from the pre-Golgi compartment (5).…”