Fig 1.
Crystal structures and dimers of CXCR4.
(a) Structure of CXCR4 in cartoon representation. The coloring of the TM is as follows: TM1 is shown in red (Ala34-Gly64), TM2 in marine blue (Met72-Ala100), TM3 in yellow (Asn106-Val139), TM4 in grey (Gln145-Phe174), TM5 in magenta (Asp193-Ser227), TM6 in dark blue (Leu238-Leu267), and TM7 in green (Cys274-Leu301). (b) Dimerization interfaces as observed in crystal structures (pdb entries 3OE9 and 3OE8 [17]).
Fig 2.
Spontaneously assembled CXCR4 dimers.
(a) Sampled CXCR4 dimer configurations in pure POPC bilayers after 3 μs (left), and in mixed POPC:cholesterol bilayers after 6 μs simulation time at 10% (middle) and 30% cholesterol content (right). The β and χ angles are defined relative to the crystal TM5,6/TM5,6 interface (central blue dots, see Methods Section for the exact definition of the angles β and χ). (b) Relative populations for the dimer configurations defined in (a) and (c). (c) Representative dimer configurations corresponding to labeled maxima A to G in (a) are shown in cylindric representation and colored equally as in Fig 1. Symmetric structures (C1 and C2 corresponding to TM1/TM5 and TM5/TM1, respectively) are distinguished by indices 1 and 2. The reference structure for all dimer configurations is indicated by green and blue axes.
Fig 3.
TM1/TM5-7 and TM1/TM1 dimer interfaces and structure alignments.
Structure alignments between spontaneously formed TM1/TM5-7 dimer (a) as well as TM1/TM1 dimer (b), obtained from a 3 μs simulation in pure POPC and backmapped to atomistic resolution (colored TM helices), and the crystal structures of TM1/TM5-7 and TM1/TM1 found in PDB-entries 3OE8 and 3OE9, respectively (light grey). The interacting residues are highlighted as sticks and labeled.
Fig 4.
Cholesterol occupation hotspots on CXCR4.
Cholesterol binding occupancy in simulations of CXCR4 dimerization at 10% cholesterol concentration. The color and thickness coded residue-resolved occupancy scale ranges from 0 to 65% time occupancy (red, thick). The inset shows a detailed view on the main cholesterol binding site at the TM1,7 interface with the main involved residues in stick representation. Additionally, a bound cholesterol molecule is depicted (gray).
Fig 5.
Cholesterol intercalation at the cholesterol-induced dimeric interface.
TM3,4/TM3,4 dimer F with two symmetrically intercalated cholesterols (shown as hotpink sticks) (structure after 100 ns atomistic MD simulation). The surrounding membrane is shown in light orange (POPC) and teal (cholesterol) sticks. In the insert, the two intercalating cholesterol molecules are highlighted together with their surroundings and water molecules (shown as spheres) forming bridges between the cholesterols and between Thr168 and cholesterol.
Table 1.
Overview over all performed coarse-grained simulations.
Table 2.
Overview over all performed atomistic simulations.
Fig 6.
Concentration of receptor monomers as a function of simulation time.
The first 500 ns were discarded for equilibration purposes.
Table 3.
Reaction rate constants, dissocation constants as well as binding free energies.
Fig 7.
Histograms of monomer lifetimes after dissociation of dimers.
Fig 8.
Relative orientation angles used to describe the orientation of the monomers in the receptor dimer.
β describes the position of monomer B with respect to monomer A, φ is the rotation of monomer B around its z-axis, χ the angle under which monomer B “sees” monomer A. The axes x and y were placed into the monomer in a way that the symmetric TM5,6/TM5,6 dimer results in β = 0 and χ = 0.
Fig 9.
Relative orientation angles for TM5/TM1 or TM1/TM5 dimers a, corresponding to β = 0°, χ = 180° or β = 180°, χ = 0°, respectively.
The symmetric TM5,6/TM5,6 dimer b binds with β = 0° and χ = 0°.
Fig 10.
Spreading of each maximum in different dimerization set-ups.
Table 4.
Diffusion constants.