Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na
-dependent uptake of ω-3 ...fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na
. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na
-bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a "trap-and-flip" mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.
Abstract only
Substrates, inhibitors, and various classes of drugs of abuse that target the human dopamine transporter (hDAT) elicit a variety of responses measurable in vitro and in vivo. ...Classification of such ligands in terms of their chemical and/or pharmacological properties has failed to produce sufficient mechanistic explanations, or hypotheses, to guide a satisfactory understanding of the nature of this diversity in the effects of hDAT ligands. With the objective of advancing the current understanding of these drugs and their mechanisms of action, we set out to investigate the role that the response of the hDAT molecule to the binding of these ligands may have in their known diverse behavioral effects. We use structurally and pharmacologically diverse ligands of hDAT (e.g., typical inhibitors, atypical inhibitors, substrates and releasers) in extensive all‐atom molecular dynamics (MD) simulations to reveal the structural and dynamic effects that characterize the response of the transporter molecule to each of them and to enable a classification of hDAT molecular responses. MD trajectories describing the time‐dependent dynamic behavior of each complex are analyzed to quantify the molecular responses of hDAT, and machine learning algorithms serve to classify each response and reveal specific dynamic features underlying the molecular mechanism of hDAT’s response to each ligand. This approach offers mechanistic insight into the role of the ligand‐determined molecular response of hDAT in determining the diversity of effects expressed in the functional outcomes.
Support or Funding Information
SB is supported by T32 “Genetic and environmental influences on addiction” (DA03980), research is supported by NIH Grant R01 DA041510 (to H.W.)
•Concepts and methodologies for the initial steps of a membrane protein setup.•Present the strengths and limitations of various software packages and tools for setup and analysis.•Highlight the open ...challenges faced by novices.•Workflow of the setup of a membrane transport protein, hDAT in complex with various stimulants.
Molecular dynamics (MD) simulations have developed into an invaluable tool in bimolecular research, due to the capability of the method in capturing molecular events and structural transitions that describe the function as well as the physiochemical properties of biomolecular systems. Due to the progressive development of more efficient algorithms, expansion of the available computational resources, as well as the emergence of more advanced methodologies, the scope of computational studies has increased vastly over time. We now have access to a multitude of online databases, software packages, larger molecular systems and novel ligands due to the phenomenon of emerging novel psychoactive substances (NPS). With so many advances in the field, it is understandable that novices will no doubt find it challenging setting up a protein-ligand system even before they run their first MD simulation. These initial steps, such as homology modelling, ligand docking, parameterization, protein preparation and membrane setup have become a fundamental part of the drug discovery pipeline, and many areas of biomolecular sciences benefit from the applications provided by these technologies. However, there still remains no standard on their usage. Therefore, our aim within this review is to provide a clear overview of a variety of concepts and methodologies to consider, providing a workflow for a case study of a membrane transport protein, the full-length human dopamine transporter (hDAT) in complex with different stimulants, where MD simulations have recently been applied successfully.
The dopamine transporter (DAT) plays a crucial role in regulating dopaminergic signaling by mediating the reuptake of dopamine (DA) from the synaptic cleft into presynaptic neurons. Dysfunction in ...DAT has been linked to various neuropsychiatric disorders, highlighting its importance in neuronal communication. Additionally, DAT is the triggering molecular target for drugs of abuse like cocaine and amphetamine, further highlighting its significance in both physiological and therapeutic contexts. Because DAT binding has been identified as the common first step in the initiation of the various behavioral effects produced by inhibitors and substrates, a central question addressed in my thesis work is the molecular basis for the various phenotypes and how ligands binding to the same canonical DAT binding site can elicit a range of different behavioral responses. Based on the structural information and mechanistic clues in the literature, I used approaches of computational biophysics starting from molecular dynamics (MD) simulations and advanced analytical tools to probe the central hypothesis of this work that the relation between different structural dynamics responses of the transporter to specific ligand classes is a mechanistic trigger for the differential pharmacological and behavioral responses that these ligands engender in the signaling cascade initiated by the transporter. My detailed findings suggest and support the mechanistic proposal that differences in structural responses of the transporter to specific ligand classes trigger the differential pharmacological and behavioral responses that these ligands engender by differentially regulating the signaling cascades initiated by the active transporter, such as in response to the binding of amphetamine which, unlike other substrates, induces reverse transport of dopamine (efflux) from inside the cell to the synapse. The conformational state of hDAT stabilized in this case leads to the dimerization of the transporter that is required for efflux. Thus, this study contributes to a deeper understanding of the molecular mechanisms governing DAT function and the way in which they can trigger various physiological consequences, paving the way for future research aimed at developing targeted interventions for neuropsychiatric disorders and substance abuse.
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•Coarse-grained modeling of mutation and binding in HIV-1 capsid.•Dynamics are robust against simulated linking of capsomers within hexamers.•Inter-hexamer linking stabilizes most ...mobile regions of capsid.
The HIV-1 capsid is a conical protein shell made up of hexamers and pentamers of the capsid protein. The capsid houses the viral genome and replication machinery, and its opening, or uncoating, within the host cell marks a critical step in the HIV-1 lifecycle. Binding of host factors such as TRIM5α and cyclophilin A (CypA) can alter the capsid’s stability, accelerating or delaying the onset of uncoating and disrupting infectivity. We employ coarse-grained computational modeling to investigate the effects of point mutations and host factor binding on HIV-1 capsid stability. We find that the largest fluctuations occur in the low-curvature regions of the capsid, and that its structural dynamics are affected by perturbations at the inter-hexamer interfaces and near the CypA binding loop, suggesting roles for these features in capsid stability. Our models show that linking capsid proteins across hexamers attenuates vibration in the low-curvature regions of the capsid, but that linking within hexamers does not. These results indicate a possible mechanism through which CypA binding alters capsid stability and highlight the utility of coarse-grained network modeling for understanding capsid mechanics.
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