Systems

Two different setups for the CG self-assembly simulations were used. A system with sixteen hSERT monomers was run in 10 repeats each lasting 30 μs and a system consisting of 64 hSERT monomers was run for 250 μs. The small setup was started with the sixteen copies of hSERT placed on a four-by-four grid using a pre-equilibrated system containing one hSERT in a membrane patch made of 99 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid molecules (Fig. 1). A 500 ns simulation with soft position restrain (100 kJ/mol/nm² in the membrane plane) on a central residue of hSERT (Leu338) was used to randomize the orientation of the sixteen copies. The large system with 64 hSERT monomers was prepared in a similar s.

For the calculations of the potential of mean force we used systems composed of a pair of hSERT molecules inserted into a rectangular box (Fig. 2A) containing ~630 POPC molecules, or a mixture of 572 POPC and 63 1-palmitoyl-2-oleoyl-PIP2 (POPIP2) molecules (9:1 ratio) and a mixture of 507 POPC and 126 CHOL molecules (4:1 ratio). The pairs of hSERTs were dimer conformations extracted from the self-assembly simulations, which were relaxed in the corresponding lipid mixture prior to US simulations. The orientation of the hSERT dimers was chosen such that their distance (the reaction coordinate) would vary along the long side of the box. A position restrain (10 kJ/mol/nm²) was applied on each hSERT molecule (backbone bead of Leu338) such that they remain close to the center of the small side of the box.

The systems studied at the atomistic resolution contained a monomeric structure of hSERT embedded into a 380 POPC lipid bilayer and two dimer conformations back-mapped from CG structures used in the calculation of the PMFs. These systems were neutralized by adding 0.2 M NaCl in the aqueous phase.