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.Fil: Friedman, Ran. Linnæus University; ArgentinaFil: Khalid, Syma. University of Southampton; Reino UnidoFil: Aponte Santamaría, Camilo. Ruprecht-Karls-Universität Heidelberg; Alemania. Universidad de los Andes; ColombiaFil: Arutyunova, Elena. University of Alberta; CanadáFil: Becker, Marlon. Westfälische Wilhelms Universität; AlemaniaFil: Boyd, Kevin J.. University of Connecticut; Estados UnidosFil: Christensen, Mikkel. University Aarhus; DinamarcaFil: Coimbra, João T. S.. Universidad de Porto; PortugalFil: Concilio, Simona. Universita di Salerno; ItaliaFil: Daday, Csaba. Heidelberg Institute for Theoretical Studies; AlemaniaFil: Eerden, Floris J. van. University of Groningen; Países BajosFil: Fernandes, Pedro A.. Universidad de Porto; PortugalFil: Gräter, Frauke. Heidelberg University; Alemania. Heidelberg Institute for Theoretical Studies; AlemaniaFil: Hakobyan, Davit. Westfälische Wilhelms Universität; AlemaniaFil: Heuer, Andreas. Westfälische Wilhelms Universität; AlemaniaFil: Karathanou, Konstantina. Freie Universität Berlin; AlemaniaFil: Keller, Fabian. Westfälische Wilhelms Universität; AlemaniaFil: Lemieux, M. Joanne. University of Alberta; CanadáFil: Marrink, Siewert J.. University of Groningen; Países BajosFil: May, Eric R.. University of Connecticut; Estados UnidosFil: Mazumdar, Antara. University of Groningen; Países BajosFil: Naftalin, Richard. Colegio Universitario de Londres; Reino UnidoFil: Pickholz, Mónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Piotto, Stefano. Universita di Salerno; ItaliaFil: Pohl, Peter. Johannes Kepler University; AustriaFil: Quinn, Peter. Colegio Universitario de Londres; Reino UnidoFil: Ramos, Maria J.. Universidad de Porto; PortugalFil: Schiøtt, Birgit. University Aarhus; DinamarcaFil: Sengupta, Durba. National Chemical Laboratory India; IndiaFil: Sessa, Lucia. Universita di Salerno; ItaliaFil: Vanni, Stefano. University Of Fribourg;Fil: Zeppelin, Talia. University Aarhus; DinamarcaFil: Zoni, Valeria. University of Fribourg; SuizaFil: Bondar, Ana-Nicoleta. Freie Universität Berlin; AlemaniaFil: Domene, Carmen. University of Oxford; Reino Unido. University of Bath; Reino Unid

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

<div><p>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.</p></div

Cholesterol density maps at the surface of the monoamine transporters.

<p>(A) hDAT overview with the 12 TM helices highlighted. The two co-crystallized CHOL molecules observed in the dDAT crystal structures are superimposed on CG hDAT for emphasizing the location of site 1 and 2 (orange sticks). (B) CHOL occupancy maps for the four CG systems: hDAT, hSERT, hNET, and dDAT in the presence of 20% cholesterol. The maps depict an occupancy level at least 3 times higher than the values corresponding to the bulk region. The 5 conserved sites are indicated. Going from left to right, the structures are shown in topview (EC) and from two different sides (separated by a 180° rotation around the membrane normal) from within the membrane plane.</p

Out-to-inward conformational transition in hDAT and hSERT.

<p>Time-resolved values of six probes monitoring the early events of the out-to-inward transition for two representative hDAT simulations (<i>AA-hDAT-w-CHOL</i>: MD5, <i>AA-hDAT-wo-CHOL</i>:MD4) and a single hSERT simulation (<i>AA-hSERT</i>: MD1) is illustrated. The TM5 RMSD, degree of helicity (%), and kink angle (ϴ) were calculated for the IC half of TM5 (residues 258–273). The “#water” is the number of water molecules within 10 Å of the Na2 site. The SASA was calculated for the IC exit path (residues F69, S72, G75, G258, S262, V266, T269, F332, G425, E428, and T432), and for T261 alone. For each probe a dashed line indicates the value observed in an IN-OCC_bound conformation for hDAT which was visually determined. The values are 5 Å for TM5 RMSD, 50% for the helicity, 20° for ϴ, 15 for #water, 220 and 50 Ų for the SASA of the IC cavity and T261, respectively. It should be noted that the hSERT simulation ran for 2 <b>μ</b>s in total, however to make comparison easier the last <b>μ</b>s of the simulation is omitted from the figure. To see the behavior of the full simulation see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005907#pcbi.1005907.s010" target="_blank">S7 Fig</a>.</p

Stability of CHOL at site 1 and 2.

<p>Stability of CHOL binding at site 1 and 2 in AA MD simulations (MD1 to 5 from top to bottom). The distance monitors the separation between the COMs of a CHOL molecule and their respective binding sites. A bound CHOL molecule is defined as when the distance is shorter than 5 Å (dashed line). Note that in MD3, CHOL at site 2 diffuses further away than the graph’s cutoff (35 Å).</p

Principal component analysis of hDAT inward-opening.

<p>(A) Porcupine plot illustrating the extreme structures of the first eigenvector extracted from a PCA of <i>AA-hDAT-wo-CHOL</i> MD4. This principal component reflects the kinking and unwinding of TM5. (B) Projection of hDAT trajectories with CHOL (<i>AA-hDAT-w-CHOL</i>) and without CHOL bound (<i>AA-hDAT-wo-CHOL</i>) onto the same principal component.</p

CHOL occupancy of site 1 and 2 as a fraction of time.

<p>CHOL occupancy of site 1 and 2 as a fraction of time.</p

Assessment of the stabilizing effects of CHOL on the out-to-inward transition of hDAT.

<p>(A) Overlay of the last frame of two representative simulations of hDAT, one with (no transition) and one without (shows transition) CHOL. TM1, TM5, and TM7 are shown in red, green, and blue, respectively, except for the EC end of TM5 which is shown in dark blue in the hDAT structure without CHOL, thus highlighting the conformational change. The two Na⁺ ions (Na1 and Na2), Cl^- (Cl), and P273 are shown. (B) Systematic comparison of repeat simulations (MD1-5) with (right) and without (left) CHOL. For each simulation the time-resolved values of six parameters are monitored: RMSD of TM5, the degree of helicity of TM5 (%), the kink angle of TM5 (ϴ), the number of water molecules within 10 Å of the Na2 site, the SASA for residues proposed to line the IC exit pathway in hSERT (F69, S72, G75, G258, S262, V266, T269, F332, G425, E428, and T432), and finally the SASA for T261. The dashed lines indicate the values observed in an IN-OCC_bound conformation of hDAT and were visually determined from <i>AA-hDAT-wo-CHOL</i> MD2-4. The values are 5 Å for TM5 RMSD, 50% for the helicity, 20° for ϴ, 15 for #water, 220 and 50 Ų for the SASA of the IC cavity and T261, respectively.</p

Sequence alignment of hDAT, hNET, hSERT and dDAT.

<p>hDAT numbering is indicated. CRAC (pink) and CARC (blue) CHOL binding motifs are highlighted in each transporter. The protein sections identified as CHOL binding sites (site 1–5) based on CG occupancy maps are highlighted using a different color for each site. For simplicity, the long EC loop 2 is omitted from the alignment. A vertical line between TM3 and TM4 indicates the location of the extracellular loop 2.</p