Inhibition of XMRV and HIV-1 proteases by pepstatin A and acetyl-pepstatin

Abstract

The kinetic properties of two classical inhibitors of aspartic proteases (PRs), pepstatin A and acetyl-pepstatin, were compared in their interactions with HIV-1 and xenotropic murine leukemia virus related virus (XMRV) PRs. Both compounds are substantially weaker inhibitors of XMRV PR than of HIV-1 PR. Previous kinetic and structural studies characterized HIV-1 PR-acetyl-pepstatin and XMRV PR-pepstatin A complexes and suggested dramatically different binding modes. Interaction energies were calculated for the possible binding modes and suggested a strong preference for the one-inhibitor binding mode for HIV-1 PR-acetyl-pepstatin and the two-inhibitor binding mode for XMRV PR-pepstatin A interactions. Comparison of the molecular models suggested that in the case of XMRV PR the relatively unfavorable interactions at S3' and the favorable interactions at S4 and S4' sites with the statine residues may shift the ground state binding towards the two-inhibitor binding mode, whereas the single molecule ground state binding of statines to the HIV-1 PR appear to be more favorable. The preferred single molecular binding to HIV-1 PR allows the formation of the transition state complex, represented by substantially better binding constants. Intriguingly, the crystal structure of the complex of acetyl-pepstatin with XMRV PR has shown a mixed type of binding: the unusual binding mode of two molecules of the inhibitor to the enzyme, in a mode very similar to the previously determined complex with pepstatin A, together with the classical binding mode found for HIV-1 PR. The structure is thus in good agreement with the very similar interaction energies calculated for the two types of binding.

Figure 1.. Structure-based sequence alignment of HIV-1, XMRV and MLV proteases.

Differing residues between XMRV and MLV proteases are indicated by arrows. Identical residues of the aligned sequences are indicated by asterisks and the binding site-forming residues are shown on gray background.

Figure 2.. Comparison of the overall structures and dimer interfaces of HIV-1 and XMRV proteases.

(A) Superposition of overall structures of HIV-1 PR (magenta) and XMRV PR (green) using ribbon/tube representation. (B) Comparison of dimer interface regions of HIV-1 PR (left) and XMRV PR (right) using ribbon/tube representation (yellow: beta-sheet, cyan: loop, magenta: alpha-helix). The N- and C-terminal ends of the monomers are indicated.

Figure 3.. Chemical structures of the inhibitors and their mode of binding to retroviral proteases.

Chemical structures of (panel A) acetyl-pepstatin and (panel B) pepstatin A. Panel C: the binding mode of acetyl-pepstatin (green) to HIV-1 PR (PDB ID: 5HVP) [13] and of pepstatin A (magenta) to XMRV PR (PDB ID: 3SM1) [15].

Figure 4.. Inhibition of the XMRV PR-mediated cleavage of a recombinant MLV Gag fragment by acetyl-pepstatin and pepstatin

A. Cleavage of the recombinant MLV Gag fragment by XMRV protease. Recombinant MLV Gag fragment was incubated for 1 h alone (lane 1) or together with XMRV PR (30 nM) in the absence of any inhibitor (lane 2) as well as in the presence of acetyl-pepstatin (3.1 μM, lane 3) or pepstatin A (28 μM, lane 4). Reactions were stopped by the addition of loading buffer and subjected to SDS-PAGE followed by Coomassie staining. Molecular masses (kDa) of the protein markers (lane M, Fermentas, SM 0431) are indicated. Arrows indicate the uncleaved recombinant protein (Δp12-CA-NC) and its fragments.

Figure 5.. Schematic representation of the binding modes of pepstatin A and acetyl-pepstatin to HIV-1 and XMRV PRs.

Single inhibitor bound-states are presented on the left-hand side, while the two inhibitor bound-states are shown on the right-hand side. Preferred binding modes (based on the crystal structures) are shown in boxes. The arrows show the direction of the inhibitors from its N-terminal end to the C-terminal end, while the circles indicate the residues of the inhibitors. Shades of the residues approximate their hydrophobicity, darker higher and brighter lower. Pepstatin A and acetyl-pepstatin molecules differ only in their N-terminal residue, thus the brighter shade indicates the acetyl group at the N-terminal end of the inhibitor, while the isovaleryl group is indicated in the same N-terminal position with the darker shade. The size of the substrate binding pockets below the dotted lines complements the size of the most preferred residues [7], dashed lines for the S4 subsites indicate that these pockets are less defined than the other ones [7]. Relatively preferred side-chain-subsite interactions are indicated by green arrows and the non-preferred ones by red arrows.

Figure 6.. Catalytic residues of XMRV PR and bound acetyl-pepstatin.

The 2Fo-Fc electron density map was contoured at 1.0 σ. Partially occupied molecules are shown in cyan for the “standard” orientation, and in yellow for the orientation resembling the mode of binding of pepstatin A. The figure was prepared with PyMol [32].

Figure 7.. Urea dissociation curve of XMRV and HIV-1 PRs.

The activity of the HIV-1 PR (solid circles) and XMRV PR (open circles) were measured at increasing urea concentration, using an HPLC detection of the substrate cleavage as described in the Materials and Methods.