Protein conformational plasticity and complex ligand-binding kinetics explored by atomistic simulations and Markov models

Abstract

Understanding the structural mechanisms of protein-ligand binding and their dependence on protein sequence and conformation is of fundamental importance for biomedical research. Here we investigate the interplay of conformational change and ligand-binding kinetics for the serine protease Trypsin and its competitive inhibitor Benzamidine with an extensive set of 150 μs molecular dynamics simulation data, analysed using a Markov state model. Seven metastable conformations with different binding pocket structures are found that interconvert at timescales of tens of microseconds. These conformations differ in their substrate-binding affinities and binding/dissociation rates. For each metastable state, corresponding solved structures of Trypsin mutants or similar serine proteases are contained in the protein data bank. Thus, our wild-type simulations explore a space of conformations that can be individually stabilized by adding ligands or making suitable changes in protein sequence. These findings provide direct evidence of conformational plasticity in receptors.

Figure 1. Apo-state structures and kinetics.

(a) Structural features, equilibrium distribution and kinetics of six unbound (apo) protein conformations. Transitions between them occur at timescales on order of tens of microseconds. The three slowest relaxation timescales and their corresponding transition process are indicated (dashed lines). The circles have an area proportional to the equilibrium probability π_i. Their respective free energy differences ΔG_b of binding a ligand to this conformation and the binding time t_bind (mean first passage time to binding) are given. The arrows indicate the transition probabilities for direct transitions between the different states (see legend). The most important structural differences concerning ligand binding are shown in b–e, and the structures are classified with respect to these features in by green/orange/red bullets in a. The structures are classified by the state of S1 or S1*: open (green circle with ‘1' or ‘1*'), half-open (orange circle) or closed (red circle) and by the S1* pocket conformational switch: favourable for binding (green circle with ‘Sw') or unfavourable for binding (red circle with ‘Sw').

Figure 2. Metastable state conformations compared with serine protease X-ray structures.

Conformations found in Trypsin wild type (coloured as in Fig. 1) are matched by crystallographic structures (grey) of other serine proteases. Similar binding site conformations are found in prostasin (blue Trypsin conformations) and the Thrombin Y225P mutant (green) as well as several other Thrombin mutants. The magenta Trypsin conformation corresponds to its wild-type structure in PDB 3PTB. The orange Trypsin wild-type conformation is similar to the X-ray structures of the Trypsin mutants S214E and S214K. The red Trypsin conformations have no equivalent crystallographic structures. Similar to the green state it has similarities to Thrombin Y225P and Kallikrein.

Figure 3. Benzamidine binding to different Trypsin conformations.

Trypsin conformations with Benzamidine-bound and the binding mode of Benzamidine. The seven conformational states shown are equal to the six apo states shown in Fig. 1, plus the yellow conformation that is only found with Benzamidine-bound. The binding pocket conformation is defined by three loops: the yellow loop (residues 187–194) with Asp189, the green loop (residues 215–221) with Trp215 and the orange loop (residues 225–230). The circles have an area proportional to the equilibrium probability of the respective conformation, given that Benzamidine is bound, π_i. Their respective relative free energies G=−k_BT ln π_i and the unbinding times t_unbind (mean first passage time to unbinding) are given. The arrows indicate the transition probabilities for direct transitions between the different states. The binding mode (pocket 1 or 1*) is indicated by the green square with ‘1' or ‘1*'.

Figure 4. Kinetic network of binding and conformational dynamics.

Kinetic network of Trypsin–Benzamidine binding and Trypsin conformational dynamics. Size of circles indicates the free energy of the states (proportional to −ln π_i). Widths of arrows indicate transition probabilities (proportional to −ln p_ij, see legend). The colours are identical to Fig. 1.

Figure 5. Binding and rebinding pathways.

Binding and rebinding pathways with probability fluxes f_ij between states i and j obtained using the transition path theory. (a) Binding pathways from the most stable unbound (apo) to the most stable bound state. Part of the mechanism is a transition from a binding-incompetent to a binding-competent structure that includes a rearrangement of biding pocket 2 shown by the green and red loops in c. (b) Rebinding pathways from a misbound structure to the most probable bound structure. The complex is most likely to first dissociate and then reassociate, indicating that conformational selection dominates the kinetics. (c) Structures of the main rebinding pathways in b.