Ultra-high resolution crystal structure of HIV-1 protease mutant reveals two binding sites for clinical inhibitor TMC114 - PubMed (original) (raw)

Ultra-high resolution crystal structure of HIV-1 protease mutant reveals two binding sites for clinical inhibitor TMC114

Andrey Y Kovalevsky et al. J Mol Biol. 2006.

Erratum in

Abstract

TMC114 (darunavir) is a promising clinical inhibitor of HIV-1 protease (PR) for treatment of drug resistant HIV/AIDS. We report the ultra-high 0.84 A resolution crystal structure of the TMC114 complex with PR containing the drug-resistant mutation V32I (PR(V32I)), and the 1.22 A resolution structure of a complex with PR(M46L). These structures show TMC114 bound at two distinct sites, one in the active-site cavity and the second on the surface of one of the flexible flaps in the PR dimer. Remarkably, TMC114 binds at these two sites simultaneously in two diastereomers related by inversion of the sulfonamide nitrogen. Moreover, the flap site is shaped to accommodate the diastereomer with the S-enantiomeric nitrogen rather than the one with the R-enantiomeric nitrogen. The existence of the second binding site and two diastereomers suggest a mechanism for the high effectiveness of TMC114 on drug-resistant HIV and the potential design of new inhibitors.

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Figures

Figure 1

Figure 1

(a) PRV32I dimer structure. Two subunits (in red and blue) are shown indicating the secondary structure. TMC114 is in ball-and-stick representation colored by atom type, and is bound in two sites. (b) and (c) The electron density (2FO-FC) for residues 55-60 in the PRV32I and PRM46L structures. Contour levels are 3.6σ for (b) and 2.4σ for (c). (d) The 2FO-FC electron density for TMC114 bound to the flap in PRV32I contoured at 1.8σ. TMC114 has 60% occupancy, while the other 40% correspond to a DMSO solvent molecule, depicted in magenta. (e) Structures of TMC114 bound in the active site cavity (_R_-enantiomer) and in the flap region (_S_-enantiomer). The moieties in the box are related by reflection in a mirror and can be obtained by inversion of the sulfonamide nitrogen.

Figure 1

Figure 1

(a) PRV32I dimer structure. Two subunits (in red and blue) are shown indicating the secondary structure. TMC114 is in ball-and-stick representation colored by atom type, and is bound in two sites. (b) and (c) The electron density (2FO-FC) for residues 55-60 in the PRV32I and PRM46L structures. Contour levels are 3.6σ for (b) and 2.4σ for (c). (d) The 2FO-FC electron density for TMC114 bound to the flap in PRV32I contoured at 1.8σ. TMC114 has 60% occupancy, while the other 40% correspond to a DMSO solvent molecule, depicted in magenta. (e) Structures of TMC114 bound in the active site cavity (_R_-enantiomer) and in the flap region (_S_-enantiomer). The moieties in the box are related by reflection in a mirror and can be obtained by inversion of the sulfonamide nitrogen.

Figure 1

Figure 1

(a) PRV32I dimer structure. Two subunits (in red and blue) are shown indicating the secondary structure. TMC114 is in ball-and-stick representation colored by atom type, and is bound in two sites. (b) and (c) The electron density (2FO-FC) for residues 55-60 in the PRV32I and PRM46L structures. Contour levels are 3.6σ for (b) and 2.4σ for (c). (d) The 2FO-FC electron density for TMC114 bound to the flap in PRV32I contoured at 1.8σ. TMC114 has 60% occupancy, while the other 40% correspond to a DMSO solvent molecule, depicted in magenta. (e) Structures of TMC114 bound in the active site cavity (_R_-enantiomer) and in the flap region (_S_-enantiomer). The moieties in the box are related by reflection in a mirror and can be obtained by inversion of the sulfonamide nitrogen.

Figure 1

Figure 1

(a) PRV32I dimer structure. Two subunits (in red and blue) are shown indicating the secondary structure. TMC114 is in ball-and-stick representation colored by atom type, and is bound in two sites. (b) and (c) The electron density (2FO-FC) for residues 55-60 in the PRV32I and PRM46L structures. Contour levels are 3.6σ for (b) and 2.4σ for (c). (d) The 2FO-FC electron density for TMC114 bound to the flap in PRV32I contoured at 1.8σ. TMC114 has 60% occupancy, while the other 40% correspond to a DMSO solvent molecule, depicted in magenta. (e) Structures of TMC114 bound in the active site cavity (_R_-enantiomer) and in the flap region (_S_-enantiomer). The moieties in the box are related by reflection in a mirror and can be obtained by inversion of the sulfonamide nitrogen.

Figure 1

Figure 1

(a) PRV32I dimer structure. Two subunits (in red and blue) are shown indicating the secondary structure. TMC114 is in ball-and-stick representation colored by atom type, and is bound in two sites. (b) and (c) The electron density (2FO-FC) for residues 55-60 in the PRV32I and PRM46L structures. Contour levels are 3.6σ for (b) and 2.4σ for (c). (d) The 2FO-FC electron density for TMC114 bound to the flap in PRV32I contoured at 1.8σ. TMC114 has 60% occupancy, while the other 40% correspond to a DMSO solvent molecule, depicted in magenta. (e) Structures of TMC114 bound in the active site cavity (_R_-enantiomer) and in the flap region (_S_-enantiomer). The moieties in the box are related by reflection in a mirror and can be obtained by inversion of the sulfonamide nitrogen.

Figure 2

Figure 2

Hydrogen bonds between the central OH group of TMC114 and the catalytic Asp25 and Asp25'. The major conformation of TMC114 is colored by atom type, and the minor conformation is green. Interatomic distances are shown in Å (a) PR-TMC114 (PDB code 1S6G); the TMC114 conformations were refined with 55% and 45% occupancies. (b) PRV32I–TMC114 and (c) PRM46L–TMC114. The 2FO-FC electron density for the active site residues Asp25 and Asp25' is shown with the contour levels of 2.2σ. The alternate conformations have occupancies of 60% and 40%.

Figure 2

Figure 2

Hydrogen bonds between the central OH group of TMC114 and the catalytic Asp25 and Asp25'. The major conformation of TMC114 is colored by atom type, and the minor conformation is green. Interatomic distances are shown in Å (a) PR-TMC114 (PDB code 1S6G); the TMC114 conformations were refined with 55% and 45% occupancies. (b) PRV32I–TMC114 and (c) PRM46L–TMC114. The 2FO-FC electron density for the active site residues Asp25 and Asp25' is shown with the contour levels of 2.2σ. The alternate conformations have occupancies of 60% and 40%.

Figure 2

Figure 2

Hydrogen bonds between the central OH group of TMC114 and the catalytic Asp25 and Asp25'. The major conformation of TMC114 is colored by atom type, and the minor conformation is green. Interatomic distances are shown in Å (a) PR-TMC114 (PDB code 1S6G); the TMC114 conformations were refined with 55% and 45% occupancies. (b) PRV32I–TMC114 and (c) PRM46L–TMC114. The 2FO-FC electron density for the active site residues Asp25 and Asp25' is shown with the contour levels of 2.2σ. The alternate conformations have occupancies of 60% and 40%.

Figure 3

Figure 3

Hydrogen bond, C-H…O and C-H…π interactions are shown in the active site cavity of PRV32I for the major conformation of TMC114 (a) and the minor conformation (b). Interactions for the alternate conformations of TMC114 in PR and PRM46L are shown in the Supplementary Material.

Figure 3

Figure 3

Hydrogen bond, C-H…O and C-H…π interactions are shown in the active site cavity of PRV32I for the major conformation of TMC114 (a) and the minor conformation (b). Interactions for the alternate conformations of TMC114 in PR and PRM46L are shown in the Supplementary Material.

Figure 4

Figure 4

(a) TMC114 at the flap binding site in PRV32I, and (b) similar view in PR. TMC114 in the PRV32I complex, and a glycerol molecule in PR-TMC114 are in a space-filling representation and colored by atom type. The protease is represented as a surface, and the residues forming the binding site are labeled. (c) Superposition of _R_-enantiomer (magenta) from the active-site cavity with the _S_-enantiomer (colored by atom type) bound in the flap site of PRV32I. The aniline moiety of the R-enantiomer (indicated by arrow) collides with the protease residues, which would prevent it from binding in the flap site. The geometry is similar in PRM46L.

Figure 4

Figure 4

(a) TMC114 at the flap binding site in PRV32I, and (b) similar view in PR. TMC114 in the PRV32I complex, and a glycerol molecule in PR-TMC114 are in a space-filling representation and colored by atom type. The protease is represented as a surface, and the residues forming the binding site are labeled. (c) Superposition of _R_-enantiomer (magenta) from the active-site cavity with the _S_-enantiomer (colored by atom type) bound in the flap site of PRV32I. The aniline moiety of the R-enantiomer (indicated by arrow) collides with the protease residues, which would prevent it from binding in the flap site. The geometry is similar in PRM46L.

Figure 4

Figure 4

(a) TMC114 at the flap binding site in PRV32I, and (b) similar view in PR. TMC114 in the PRV32I complex, and a glycerol molecule in PR-TMC114 are in a space-filling representation and colored by atom type. The protease is represented as a surface, and the residues forming the binding site are labeled. (c) Superposition of _R_-enantiomer (magenta) from the active-site cavity with the _S_-enantiomer (colored by atom type) bound in the flap site of PRV32I. The aniline moiety of the R-enantiomer (indicated by arrow) collides with the protease residues, which would prevent it from binding in the flap site. The geometry is similar in PRM46L.

Figure 5

Figure 5

. (a) Hydrogen bond network and C-H…O interactions of TMC114 bound in the flap site of PRV32I. Hydrogen bonds are colored in red, C-H…O contacts are black, and distances are in Å The interactions in the PRM46L complex are very similar. (b) TMC114 bound to the surface site is surrounded by four protein molecules. The asymmetric unit consists of PRV32I (blue) and two inhibitor molecules shown in yellow ball-and-stick representations. The symmetry related protease molecules are in cyan, orange and magenta.

Figure 5

Figure 5

. (a) Hydrogen bond network and C-H…O interactions of TMC114 bound in the flap site of PRV32I. Hydrogen bonds are colored in red, C-H…O contacts are black, and distances are in Å The interactions in the PRM46L complex are very similar. (b) TMC114 bound to the surface site is surrounded by four protein molecules. The asymmetric unit consists of PRV32I (blue) and two inhibitor molecules shown in yellow ball-and-stick representations. The symmetry related protease molecules are in cyan, orange and magenta.

Figure 6

Figure 6

(a) The superposition of the PRV32I and PRM46L structures onto the wild type PR. (b) The residues (labeled) of the flap binding site for TMC114 have the largest differences. PR is colored by atom type, while PRV32I and PRM46L are colored in magenta and cyan, respectively. The atomic shifts (Å) are indicated by dashed arrows.

Figure 6

Figure 6

(a) The superposition of the PRV32I and PRM46L structures onto the wild type PR. (b) The residues (labeled) of the flap binding site for TMC114 have the largest differences. PR is colored by atom type, while PRV32I and PRM46L are colored in magenta and cyan, respectively. The atomic shifts (Å) are indicated by dashed arrows.

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