Flap Dynamics in Pepsin-Like Aspartic Proteases: A Computational Perspective Using Plasmepsin-II and BACE-1 as Model Systems

Abstract

The flexibility of β hairpin structure known as the flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequential similarity. In this study, we have used apo Plm-II and BACE-1 as model systems. In the apo form of the proteases, a conserved tyrosine residue in the flap region remains in a dynamic equilibrium between the normal and flipped states through rotation of the χ₁ and χ₂ angles. Independent MD simulations of Plm-II and BACE-1 remained stuck either in the normal or flipped state. Metadynamics simulations using side-chain torsion angles (χ₁ and χ₂ of tyrosine) as collective variables sampled the transition between the normal and flipped states. Qualitatively, the two states were predicted to be equally populated. The normal and flipped states were stabilized by H-bond interactions to a tryptophan residue and to the catalytic aspartate, respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine, reduced the flexibility of the flap and resulted in a flap collapse (flap loses flexibility and remains stuck in a particular state). This is in accordance with previous experimental studies, which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Our results suggest that the ring flipping associated with the tyrosine side-chain is the key order parameter that governs flap dynamics and opening of the binding pocket in most pepsin-like aspartic proteases.

Figure 1

Flap (orange) and coil (blue) region of pepsin-like aspartic protease (A) and location of the conserved tyrosine (Tyr) present in the flap (B). Difference in flap conformation in crystal and open state (C).

Figure 2

Location of the Tyr,Trp,Asp triad in a typical pepsin-like aspartic protease, Plm-II (A). Definition of the χ₁ and χ₂ angles of Tyr (B). Typical distribution of the χ₁ angle (C), where N and F denote the normal and flipped states, respectively.

Figure 3

H-bond interactions involving Tyr-77 in Plm-II, divided into those typical for the normal state (upper panel) and flipped state (lower panel).

Figure 4

Apparent free-energy surfaces (with respect to the Tyr-77 torsions) for each of the four independent MD simulations of Plm-II (denoted as MD 1–4) with the TIP3P water model and from the combined Torsion-Metad simulations (denoted as Metad). The normal (N) and flipped (F) states are marked on the χ₁ axis.

Figure 5

Free-energy surfaces for Plm-II reweighted on χ₁ and H-bond distances to Trp-41 (upper panel) and Asp-34 (lower panel) for the MD (four runs combined) and Torsion-Metad simulations, respectively. Representative structures corresponding to the two H-bonds are also shown.

Figure 6

Apparent free-energy surfaces of BACE-1 from MD and Torsion-Metad simulations starting from the SN, SO, and SSO structures, respectively. Independent MD simulations starting from the SN or SO structures only sampled the normal (N) state ( rad, whereas simulations starting from the SSO structure only sampled the flipped (F) state. Torsion-Metad simulations starting from the SN, SO, or SSO structures sampled both the N and F states.

Figure 7

Free-energy surface for the combined Torsion-Metad simulations on BACE-1 reweighted on χ₁ and different H-bond distances (Trp-76, Ser-35, Asp-32, and Lys-107). The corresponding free-energy surfaces for the individual runs (starting with the SN, SO, and SSO conformations) can be found in the Supporting Information.

Figure 8

Apparent free-energy surfaces for Plm-II reweighted on χ₁ and DIST2, showing sampling of overall flap flexibility during MD (denoted as MD1–4) and Torsion-Metad simulations. N and F denote the normal and flipped states of Tyr-77, respectively.

Figure 9

Free energy surface reweighted on χ₁ and DIST2 in MD and Torsion-Metad simulations starting with SN, SO, and SSO conformations. N and F denote the normal and flipped states of Tyr-71.

Figure 10

Time evolution of the unbinding CV (distance between the center of mass of the active site residues and ligand heavy atoms) during the metadynamics simulation (A). Time evolution of DIST2 showing the extent of flap opening during ligand exit (B). The stability of the ligand in the active site has been highlighted in Figure S23.

Figure 11

Free-energy surface projected on DIST2 and DIST3 in the case of apo Plm-II (A and B) and BACE-1 (C and D) with Ala mutation. For comparison, the wild type Plm-II (E) and BACE-1 (F) are also shown. Projection on DIST2 highlights that the flap loses its flexibility in the mutant protein.

Figure 12

Unified mechanism of conformational dynamics in pepsin-like aspartic proteases: Three different conformational states associated with side-chain flexibility of Tyr in pepsin-like aspartic proteases. The normal and flipped states are typically stabilized by H-bonds to Trp and Asp. C is the solvent-exposed subspace which consists of several rotameric conformations (C1, C2, C3, C4, etc.) of Tyr. P1 to P10 denotes crystal structures of human cathepsin-D (PDB: 1LYA), cathepsin-E (PDB: 1TZS), BACE-2 (PDB: 3ZKQ), Plm-V (PDB: 4ZL4), bovine chymosin (PDB: 4AUC), human pepsin (PDB: 3UTL), candidapepsin (PDB: 2QZW), human renin (PDB: 5SY2), Plm-I (PDB: 3QS1), and Plm-IV (PDB: 1LS5) respectively. All structures (except Plm-V) possessed conserved Tyr, Trp, and Asp residues similar to Plm-II and BACE-1. Plm-V is missing the conserved Trp residue.

Figure 13

Orientation of tyrosine in crystal structures of Plm-II [PDB: 2BJU (blue), 2IGY (magenta), and 4Z22 (green)] and bovine chymosin protease [PDB: 1CMS (gray)].