Figure 1.
The open (PDB ID: 2LAO) and closed (PDB ID: 1LAF) states of the Lysine-, Arginine-, Ornithine-binding (LAO) Protein.
The ligand, Arginine, is shown in red.
Figure 2.
The bound state of our MSM for LAO binding (which is also the most populated state, having an equilibrium population of 74.9%).
(a) A snapshot from our simulations (red) achieves a 1.2 Å RMSD to the X-ray bound state (blue, PDB ID: 1LAF). The RMSD is computed from the protein Cα atoms that are within 8 Å to the center of mass of the ligand in the X-ray bound state (Residues 9–15, 17–19, 30, 50–53, 55–56, 67–74, 77, 83, 88, 90–92, 117–124, 141–143, 159–162, 164, 190–191 and 194–196). If all-protein Cα atoms are included the RMSD is 1.8 Å. (b) Free energy plot of the protein opening angle versus twisting angle. The bin size is (5°, 5°), and the interval between two adjacent contour levels is 0.5 KT. The green and blue crosses correspond to X-ray structures of the bound and apo conformations respectively. (c) Free energy plot of the opening angle versus the distance between the ligand and the binding site. The bin size is (1.5 Å, 5°), and interval between contour levels is 0.5 KT.
Figure 3.
Superposition of the 10 highest flux pathways from the unbound states to the bound state.
The flux was calculated using a greedy backtracking algorithm [31], [66] applied to a 54-state MSM generated with the SHC algorithm [30]. These pathways account for 35% of the total flux from unbound states to the bound state. The arrow sizes are proportional to the interstate flux. State numbers and their equilibrium population calculated from MSM are also shown. The conformational selection and induced pathways from the unbound states to the encounter complex state is shown in green and grey arrows respectively.
Figure 4.
Superposition of the 10 highest flux pathways from the unbound states to the bound state as in Fig. 2A but with representative structures replaced with free energy plots of the protein opening angle versus twisting angle.
The green and blue crosses correspond to X-ray structures of the bound and apo conformations respectively. The bin size is (5°, 5°), and the interval between contour levels is 0.5 KT (same as Fig. 2b). The conformational selection and induced pathways from the unbound states to the encounter complex state is shown in green and grey arrows respectively.
Figure 5.
Structural comparisons between encounter complex and X-ray apo (or open) and bound (or closed) structures for the LAO protein.
The X-ray apo (PDB ID: 2LAO) and bound structure (PDB ID: 1LAF) are shown in green and light blue respectively. Three representative conformations from the encounter complex state are superimposed and shown in red. These three conformations are representative of 10,000 randomly selected conformations from the encounter complex state (i.e. they have the smallest protein Cα RMSD to all the rest of the randomly selected conformations and are, therefore, the most central/typical of the state). In the right panel, the open, closed X-ray structures are overlaid with one of the representative conformations from the encounter complex state.
Figure 6.
Overlay of free energy plots of the protein opening angle versus twisting angle for the encounter complex (red) and bound state (blue).
Green dots correspond to where transitions from the encounter complex to the bound state occur. Blue dots correspond to where transitions into the encounter complex from other states occur. Only transitions without re-crossing are counted (minimum residence time in the final state after the transition is 6 ns).
Figure 7.
A schematic diagram describing the proposed two-step binding mechanism for proteins in steric occlusion of the direct binding of the ligands.
The first step is the transition from the apo to the encounter complex state. In this step, multiple pathways exist where both conformational selection and the induced fit mechanisms play important roles. The second step is the transition from the encounter complex to the bound state, where the induced fit mechanism is adopted.