Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.
Plasma membranes
(PMs) contain hundreds of different lipid species
that contribute differently to overall bilayer properties. By modulation
of these properties, membrane protein function can be affected. Furthermore,
inhomogeneous lipid mixing and domains of lipid enrichment/depletion
can sort proteins and provide optimal local environments. Recent coarse-grained
(CG) Martini molecular dynamics efforts have provided glimpses into
lipid organization of different PMs: an “Average” and
a “Brain” PM. Their high complexity and large size require
long simulations (∼80 μs) for proper sampling. Thus,
these simulations are computationally taxing. This level of complexity
is beyond the possibilities of all-atom simulations, raising the question—what
complexity is needed for “realistic” bilayer properties?
We constructed CG Martini PM models of varying complexity (63 down
to 8 different lipids). Lipid tail saturations and headgroup combinations
were kept as consistent as possible for the “tissues’”
(Average/Brain) at three levels of compositional complexity. For each
system, we analyzed membrane properties to evaluate which features
can be retained at lower complexity and validate eight-component bilayers
that can act as reliable mimetics for Average or Brain PMs. Systems
of reduced complexity deliver a more robust and malleable tool for
computational membrane studies and allow for equivalent all-atom simulations
and experiments.
The oligomeric state of membrane proteins has recently emerged in many cases as having an effect on their function. However, the intrinsic dynamics of their spatial organization in cells and model systems makes it challenging to characterize. Here we use molecular dynamics (MD) simulations at multiple resolutions to determine the dimer conformation of the human serotonin transporter (hSERT). From self-assembly simulations we predict dimer candidates and subsequently quantify their relative strength. We use umbrella sampling (US) replica exchange MD simulations for which we present extensive analysis of their efficiency and improved sampling compared to regular US MD simulations. The data shows that the most stable hSERT dimer interface is symmetrical and involves transmembrane helix 12 (TM12), similar to the crystal structure of the bacterial homologue LeuT, but with a slightly different orientation. We also describe the supramolecular organization of hSERT from a 250 μs self-assembly simulation. Finally, the effects of the presence of phosphatidylinositol bisphosphate or cholesterol in the membrane model has been quantified for the TM12-TM12 predicted interface. Collectively, the presented data bring new insight to the area of protein and lipid interplay in biological membranes.
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