12 research outputs found
Cholesterol Modulates the Structure, Binding Modes, and Energetics of CaveolināMembrane Interactions
Caveolin-1 (cav-1) is an important membrane protein that
plays
a vital role in cellular signaling and trafficking by organizing membrane
domains. The peptide interacts with cholesterol-rich membranes and
induces large morphological changes in them, forming microdomains
such as caveolae. Here, we use coarse-grain molecular dynamics simulations
to study the interaction of cav-1 peptides with several model bilayer
systems mimicking biological scenarios, such as cholesterol-rich domains,
cholesterol-depleted domains, and unsaturated lipid domains. We show
that cholesterol modulates the depth as well as orientation of cav-1
binding to membranes. Furthermore, the presence of cholesterol stabilizes
more open conformations of cav-1, and we speculate that the binding
modes and open conformations could be responsible for inducing morphological
changes in the bilayer. We also calculated the partitioning free energy
to different bilayers and show that binding to cholesterol-rich bilayers
is more favorable than cholesterol-depleted bilayers and the binding
to unsaturated bilayers is the least favorable. Binding to cholesterol-rich
bilayers also changes the pressure profile of the bilayer to which
it is bound and thereby affects the local spontaneous curvature. Our
results highlight molecular details of proteinālipid interplay
and provide new insights into the effects of cav-1 in tuning the morphology
of cholesterol-rich membranes
Identification of Cholesterol Binding Sites in the Serotonin<sub>1A</sub> Receptor
The serotonin<sub>1A</sub> receptor is a representative
member
of the G protein-coupled receptor (GPCR) superfamily and serves as
an important drug target in the development of therapeutic agents
for neuropsychiatric disorders. Previous work has shown the requirement
of membrane cholesterol in the organization, dynamics, and function
of the serotonin<sub>1A</sub> receptor. We show here that membrane
cholesterol binds preferentially to certain sites on the serotonin<sub>1A</sub> receptor by performing multiple, long time scale MARTINI
coarse-grain molecular dynamics simulations. Interestingly, our results
identify the highly conserved cholesterol recognition/interaction
amino acid consensus (CRAC) motif on transmembrane helix V as one
of the sites with high cholesterol occupancy, thereby confirming its
role as a putative cholesterol binding motif. These results represent
the first direct evidence for membrane cholesterol binding to specific
sites on the serotonin<sub>1A</sub> receptor and represent an important
step in our overall understanding of GPCR function in health and disease
Estimating the Lipophobic Contributions in Model Membranes
The
insertion and association of membrane proteins is critical
in several cellular processes. These processes were thought to be
protein-driven, but increasing evidence points toward an important
role of the lipid bilayer. The lipid-mediated contribution has been
shown to be important in the association of membrane peptides, but
the corresponding ālipophobicā component has not been
directly estimated. Here, we calculate the free energy of insertion
for transmembrane peptides and estimate the lipophobic component from
the cost of cavity formation. The free-energy calculations were performed
using the coarse-grain Martini force field, which has been successful
in predicting membrane protein interactions. As expected, the charged
moieties have the least favorable free energy of insertion and the
highest cost of cavity formation. A length dependence was observed
in polyalanine peptides with the lipid-mediated component increasing
nonlinearly with peptide length. Membrane fluidity was tested by varying
the temperature, and opposing effects were observed for short and
long peptides. The dependence of the lipid-mediated effects on peptide
length and temperature was not uniform and gives valuable insight
into the anisotropic nature of the membrane. The results are an important
step in estimating membrane effects in protein insertion and association
Structural characterization of the Ī²<sub>2</sub>AR variants.
<p>All atom protein RMSD of (A) Arg and (B) Gly variants of Ī²2AR with respect to the first frame of the production run. All atom RMSD of the N-terminal residues 1 to 29 of (C) Arg and (D) Gly variants of Ī²2AR. RMSF profile of the N-terminal C-alpha atoms of (E) Arg and (F) Gly variants. For each plot, the blue line indicates the first simulation, the red indicates the second and the green line indicates the third simulation.</p
Contact maps of Arg and Gly variants of Ī²<sub>2</sub>AR.
<p>Contacts between the N-terminal region of (A) Arg and (B) Gly variants and the rest of the receptor were computed from the average distance over simulation time. Average distances that were less than or equal to 1 nm were plotted. A color bar is indicated in which white indicates no-contacts and black indicates a close contact.</p
Secondary structure of the N-terminal region of the Arg and Gly variants of Ī²<sub>2</sub>AR.
<p>Panels A, B and C represent simulation number 1, 2 and 3 of the Arg variant and panels D, E and F represent simulation number 1, 2 and 3 of the Gly variant, respectively. Turns are indicated by cyan color, 3ā10 helices are indicated by blue, alpha helices are indicated by pink, isolated bridges are indicated by mustard and extended configuration is indicated by yellow color.</p
Characterization of the ligand binding site of Ī²<sub>2</sub>AR variants.
<p>(A) Top-view of the Ī²<sub>2</sub>AR represented as ribbons. The residues (113, 203, 289 and 312) that define the topology of the binding site are represented as licorice and the distances between them are indicated by lines. (B) Average distance between residues 289 and 203 for the Arg (red) and Gly (green) variants. (C) Average distance between residues 312 and 203 for the Arg (red) and Gly (green) variants. Docking of carazolol to (D) the 2RH1 structure, (E) Arg variant and (F) Gly variant. The crystal structure pose of carazolol is colored yellow, and the docked pose is colored purple.</p
Vestibules of ligand entry in Ī²<sub>2</sub>AR variants.
<p>Grids representing vestibule openings for (A) vestibule1 and (B) vestibule 2 of Ī²<sub>2</sub>AR are shown in yellow. Residues defining the vestibules are shown in surface representation while the rest of the receptor is rendered as ribbons and colored blue. Panel C and D represent volumes (in Ć
<sup>3</sup>) of the non-occluded grid of vestibule 1 for the Arg and Gly variants, respectively. Panel E and F represent volumes (in Ć
<sup>3</sup>) of the non-occluded grid of vestibule 2 for the Arg and Gly variants, respectively. For each plot, the blue line indicates the first simulation, the red indicates the second and the green line indicates the third simulation.</p
Molecular view of the sequence of events of the leaky fusogenic action of cyclic peptides.
<p>A. Initial simulation setup with peptides placed between two bilayers. B. Bridging of proximal leaflets of the two bilayers by BPC194. C. Lipid bulging caused by the action of peptides associated with the bilayers. D. Pre-stalk intermediate accompanied by disordered toroidal pore. E. Close-up of the bridging peptides. F. Close-up of the stalk-pore complex. GāJ. Splaying of a lipid during the course of a simulation. The peptides are depicted in pink, the phosphorous atoms in yellow and green respectively and the lipid chains in grey. The water is not shown for clarity. In panel F, the water molecules within the pore in one of the bilayers are shown in blue. The other pore cannot be seen in the zoom-in but is visible in panel D.</p
Simultaneous pore formation and fusion activity of BPC194.
<p>A: The normalized concentration of dextran inside the liposomes, C<sub>av</sub>, (filled circles) and the normalized intensity of membrane-associated DiD per liposome (empty squares) at different P/L ratios. B: Confocal images of the lipid vesicles in the DiD and dextran detection channel at three different P/L ratios; Ī±, P/Lā=ā0; Ī², P/Lā=ā0.1; and Ī³, P/Lā=ā0.3. C: Positive-FRET upon peptide addition. The emission of Rhodamine increases due to vesicle fusion. Inset: Controls done with the āinactiveā linear analog of BPC194, that is, BPC193 at the same peptide concentrations. D: Negative-FRET upon peptide addition. The emission of NBD increases due to a decrease in FRET efficiency as a result of vesicle fusion. E. Quantification of fusion at different P/L ratios and at two different lipid compositions, 125 ĀµM (full circles) and 250 ĀµM (empty squares). D. Representative cryo-TEM micrographs of DOPG vesicles without peptide (control) and with BPC194 or the linear analog BPC193.</p