Effect of palmitoylation on the dimer formation of the human dopamine transporter.

The human dopamine transporter (hDAT) is one in three members of the monoamine transporter family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on which dimeric and higher order oligomeric states are accessible and energetically favorable. In this work, we present several probable dimer conformations using computational coarse-grained self-assembly simulations and assess the relative stability of the different dimer conformations using umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group (palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12 in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests similar dimer conformations across these protein family.

Capturing Biologically Complex Tissue-Specific Membranes at Different Levels of Compositional Complexity.

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.

Substrate and inhibitor binding to the serotonin transporter: Insights from computational, crystallographic, and functional studies.

The serotonin transporter (SERT) belongs to the monoamine transporter family, which also includes the dopamine and norepinephrine transporters. SERT is essential for regulating serotonergic signaling by the reuptake of serotonin from the synaptic cleft back into the presynaptic neuron. Dysregulation of SERT has been implicated in several major psychiatric disorders such as major depressive disorder (MDD). MDD was among the top five leading causes of years lived with disease in 2016 and is characterized as a major global burden. Several drugs have been developed to target SERT for use in the treatment of MDD, and their respective binding modes and locations within SERT have been studied. The elucidation of the first structure of a bacterial SERT homologue in 2005 has accelerated crystallographic, computational, and functional studies to further elucidate drug binding and method of action in SERT. Herein, we aim to highlight and compare these studies with an emphasis on what the different experimental methods conclude on substrate and inhibitor binding modes, and the potential caveats of using the different types of studies are discussed. We focus this review on the binding of cognate substrate and drugs belonging to the different families of antidepressants, including tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, and multimodal drugs, as well as illicit drugs such as cocaine, amphetamines, and ibogaine. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Molecular modeling of neurological membrane proteins - from binding sites to synapses.

The field of molecular mechanics studies of proteins has developed enormously since its origin in the 1970's, and many applications and methodologies have branched from the original idea of the force field. The applications of such methodologies are far spread and commonplace in neuroscience research today. In this mini-review, we outline the main methodologies applied when studying events ranging from ligands binding within small binding sites, through overall large-scale conformational changes, to the even larger-scale oligomerization events of neurological membrane proteins. The limitations and caveats of the methods are discussed, while examples of recent applications are described and their implications discussed. We have chosen to focus on the monoamine transporters throughout, with a few examples from neurological membrane proteins such as ionotropic and metabotropic neurotransmitter receptors.

Understanding Conformational Dynamics of Complex Lipid Mixtures Relevant to Biology.

This is a perspective article entitled "Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology" which is following a CECAM meeting with the same name.

Dimer Interface of the Human Serotonin Transporter and Effect of the Membrane Composition.

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.

A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition.

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.

Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors.

BACKGROUND: Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor ß, a folate/antifolate transporter. METHODS: We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR-TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. RESULTS: Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures. CONCLUSIONS: The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents. GENERAL SIGNIFICANCE: Apart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.