Fig. 1.
A. The core and the gate domains as suggested by Lu et al. [20] are shown in grey and cyan respectively. B. Snapshot from the end of one of the UraA-AT simulations. The same colors for UraA as in A are used. The POPE, POPG and CL are shown in blue, red and green respectively. The water solvent is shown in red. C. Sequence of UraA. The secondary structure is shown below the sequence. The same colors for the core and the gate domains as in A are used.
Table 1.
Summary of the principal simulations.
Fig. 2.
Interactions of UraA with lipids.
A. Occupancy plots showing the probability of occurrence of cardiolipin (CL; green) and of the POPG (red) lipids around UraA. The occupancy was calculated as the average over all repeat coarse-grained simulations of the wild type UraA (UraA-CG in Table 1). Three different views are shown: a side view (left) showing the three CL binding sites, a cytosolic view facing the inner leaflet (middle) showing CL sites 1 and 2, and an extracellular view facing the outer leaflet of the bilayer (right) showing CL site 3. The distribution of all lipids separately is shown in S8 Fig. B. Normalized average number of contacts (using a cut-off distance of 7 Å for POPE and POPG and 8 Å for CL molecules) between the UraA and the head groups of POPG and CL lipids in the bilayer (across all repeats of the extended UraA-CG simulations; see Table 1). For the normalization, the number of contacts of a residue with a lipid type was divided by the number of lipids, the number of frames and the ratio of cutoff volumes. C. The number of contacts from one of the CG-MD simulations was mapped on the UraA crystal structure. Blue indicates a low number, white indicates a medium number and red a large number of contacts.
Fig. 3.
Atomistic simulations of UraA in a 75% POPE/ 20% POPG/ 5% CL bilayer. Snapshots from the specific interactions between the CL and UraA are shown for the three CL binding sites (shown in green). The protein is represented as grey ribbons. The positively charged residues that form the main interactions with the CL in each CL binding site are shown in magenta. CL is shown in VDW format. Note that for this Figure we show snapshots with only CL lipids bound in the CL binding sites. As detailed in S4 Fig. and S6 Fig. and the in the text, POPG and POPE molecules to a lesser extent can also associate with the CL binding sites.
Fig. 4.
Interactions between the lipids and CL binding site 1.
A. Histograms of binding event durations (defined as the time for which CL binding site 1 was continuously occupied by a lipid; see Methods for details). B. Interactions of UraA R299 residue with the POPG and CL molecules as a function of the simulation time for one of the CG simulations with the wild type UraA (10 μs simulation; UraA-CG in Table 1). An occupancy index of 1 is used when the lipids are in contact with the protein and 0 when the lipids are not in contact. See S2 Fig. for the same analysis for all residues of UraA. C. The 10 longest stretches of continuous interactions between the different lipid types and CL binding sites 1. The same analysis for the other CL binding sites is shown in S3 Fig.
Fig. 5.
Mutations on the CL binding sites.
Occupancy plots for the location of cardiolipins (CL; green) around UraA. The occupancy was calculated as the average over the multiple coarse-grained simulations of the wild type and the mutated forms of UraA (Mut 1: UraAmut-1-CG, Mut 2: UraAmut-2-CG and Mut 3: UraAmut-3-CG; see Table 1).
Fig. 6.
A. Alignment of the core domain of the final snapshot of one of the atomistic simulations (UraA-AT) with the core domain of the crystal structure. B. Ribbon structure of UraA showing the location of the bound uracil (green) and NG detergent (orange). C,D. Pore lining surfaces of the proteins aligned in A. Red is used when the pore radius is lower than 1.15 Å, green when the pore radius is between 1.15 and 2.3 Å and blue when the pore radius is larger than 2.3 Å. Note the loss of water cavity (blue) from the inward-facing passage during the simulation to produce a closed conformational state of UraA.