Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

Figure 1

One of the two trimers in the asymmetric unit of HTLV-1 PR in complex with inhibitor KNI-10562. The trimer consists of three homodimers, with each monomer colored separately. The AB dimer is colored green and yellow the CD dimer, blue and magenta and the EF dimer, cyan and pink, respectively. The inhibitor molecules are shown in ball-and-stick representation and are colored orange for their principal orientation, whereas the alternative conformation of the inhibitor in the CD dimer is gray. The stands representing the β-hairpin called the flap are omitted, in order to visualize the inhibitors more clearly. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and green, respectively.

Figure 2

The structure of inhibitor KNI-10562. A) A difference omit map of inhibitor KNI-10562 in dimer AB contoured at 3σ. Oxygen, nitrogen and sulfur atoms are colored in red, blue and yellow, respectively. B) Superposition of the KNI-10562 inhibitor bound to six crystallographically independent protease dimers, all oriented in the same way. Inhibitors bound to dimers AB (green), EF (yellow), and KL (cyan) are all in a single conformation and are shown in thick stick models. Inhibitors bound to dimer CD (slate), GH (gray) and IJ (pink) with alternative orientation are shown in thin stick models. C) Alternative conformation of the inhibitor bound in the CD, GH and IJ dimer. Inhibitors bound to dimer CD (green), GH (yellow) and IJ (cyan) are shown in thick and thin stick models for the principal and alternative orientations, respectively.

Figure 3

Superposition of the structures of the inhibitors from the KNI series and their comparison with the statine-based inhibitor. A) Superposition of the inhibitors. KNI-10562 is green, KNI-10673 yellow, KNI-10681 slate, KNI-10683 pink, and KNI-10729 pink. B) Superposition of the KNI-10562 inhibitor with the statine-based inhibitor. KNI-10562 is yellow and the statine inhibitor is gray.

Figure 4

Hydrogen-bonded interactions between HTLV-1 PR and the inhibitors. A) The binding site of KNI-10562. The inhibitor is shown in yellow sticks, whereas the residues of the enzyme are shown in ball-and-stick representation (molecule A: green, molecule B: cyan). B) Statine-based inhibitor bound to HTLV-1 PR. The inhibitor is shown as gray sticks. Hydrogen bonds are shown in black dashes.

Figure 5

Hydrophobic interactions between HTLV-1 PR and the inhibitors. A) Interactions of KNI-10562. The inhibitor is shown with yellow sticks representing the carbon atoms, and red and blue sticks corresponding to oxygen and nitrogen, respectively. The model is covered with transparent spheres in their respective colors. The residues of the enzyme are also shown as sticks covered with transparent spheres (molecule A: green, molecule B: cyan). B) Interactions of the statine-based inhibitor, with its carbons colored gray.

Figure 6

Dual conformation of the loop 91–99 in HTLV-1 PR. The superimposed fragments of the loop comprising residues 94–98 are shown in ribbon representation. Five out of six loops with conformation 1 of the complex with the statine-based inhibitor are shown in green, whereas the sixth loop with conformation 2 is shown in red. Loops with conformation 2 in the complexes with other inhibitors are shown in pale pink, purple, yellow, magenta and cyan for KNI-10729, -10562, -10683, -10681 and -10673, respectively. The side chains of the residues of the loops are shown in stick representation.

Figure 7

Concerted conformational changes in the flaps and loops 91–99 induced by the interactions with the inhibitors. The structures of all complexes are superimposed on the basis of their Cα coordinates. AB dimers are used for the complexes with the statine-based inhibitor and KNI-10562. The flaps are shown as ribbons, fragments of loop 91–99 comprising residues 94–98 and the side chains of the flaps are shown in stick representation. The color scheme is the same as in Fig. 6. A) A view of the active sites of the enzymes interacting with the C-terminal half of the statine-based inhibitor (shown as sticks), with the KNI inhibitors shown in thin lines. B) Superimposed fragments of the active sites of the all inhibitor complexes interacting with the N-terminal halves of the inhibitors (shown as sticks).

Figure 8

Two water molecules (shown as green balls) found between the tips of the flaps of monomers A and B in the complex with the statine-containing inhibitor. The superimposed flaps from all inhibitor complexes are shown as ribbons, whereas the inhibitors are shown as sticks. The color scheme is the same as in Fig. 6.

Figure 9

A hydrogen bonded network between the flaps and the fragment of the loop 91–99. Superimposed fragments of the complexes with the statine-based inhibitor (green) and KNI-10562 (cyan) are shown as sticks, whereas the two water molecules are shown as balls. Hydrogen bonds are shown as dashed lines.