PDBsum entry 3bvb

Hydrolase

PDB id: 3bvb

Contents

Protein chains

99 a.a. *

Ligands

017

GOL

Metals

_NA

_CL

Waters ×196

* Residue conservation analysis

PDB id:

3bvb

Name:

Hydrolase

Title:

Cystal structure of HIV-1 active site mutant d25n and inhibitor darunavir

Structure:

Protease (retropepsin). Chain: a, b. Synonym: (pr). Engineered: yes. Mutation: yes

Source:

Human immunodeficiency virus 1. Organism_taxid: 11676. Gene: gag-pol. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.30Å R-factor: 0.160 R-free: 0.210

Authors:

F.Liu,I.T.Weber

Key ref:

J.M.Sayer et al. (2008). Effect of the active site D25N mutation on the structure, stability, and ligand binding of the mature HIV-1 protease. J Biol Chem, 283, 13459-13470. PubMed id: 18281688 DOI: 10.1074/jbc.M708506200

Date:

05-Jan-08 Release date: 01-Apr-08

Protein chains

P03367  (POL_HV1BR) -  Gag-Pol polyprotein from Human immunodeficiency virus type 1 group M subtype B (isolate BRU/LAI)

Seq: 1447 a.a.

Struc: 99 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.7.7.49 - RNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 2: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 3: E.C.3.1.13.2 - exoribonuclease H.
• Reaction: Exonucleolytic cleavage to 5'-phosphomonoester oligonucleotides in both 5'- to 3'- and 3'- to 5'-directions.
• Enzyme class 4: E.C.3.1.26.13 - retroviral ribonuclease H.
• Enzyme class 5: E.C.3.4.23.16 - HIV-1 retropepsin.
• Reaction: Specific for a P1 residue that is hydrophobic, and P1' variable, but often Pro.

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M708506200

J Biol Chem 283:13459-13470 (2008)

PubMed id: 18281688

Effect of the active site D25N mutation on the structure, stability, and ligand binding of the mature HIV-1 protease.

J.M.Sayer, F.Liu, R.Ishima, I.T.Weber, J.M.Louis.

ABSTRACT

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR(D25N)) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 +/- 0.09 microm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR(D25N) in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 degrees C, respectively. Only minimal differences are observed in high resolution crystal structures of PR(D25N) complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH(2) (RPB), as compared with PR.DRV and PR.RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T(m) is smaller for PR(D25N) (6.2 degrees C) than for PR (8.7 degrees C). The T(m) of PR(D25N).DRV increases by only 3 degrees C relative to free PR(D25N), as compared with a 22 degrees C increase for PR.DRV, and the mutation increases the ligand dissociation constant of PR(D25N).DRV by a factor of approximately 10(6) relative to PR.DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

Selected figure(s)

Figure 5.
Comparison of the crystal structures of PR (red)- and PR[D25N] (green)-inhibitor complexes. Tube representations of PR bound to DRV (A) or RPB (B) superimposed on PR[D25N] bound to DRV (A) or RPB (B) ranging from 1.05- to 1.4-Å resolution. Inhibitors, DRV and RPB, and the active site residue 25 are shown as stick models, and the terminal residues are indicated. The location of a central motif consisting of a hydroxyl group in DRV that can hydrogen bond to the catalytic Asp-25 is indicated by the black arrow.

Figure 6.
Comparison of the inhibitor structures bound to either PR (red or black) or PR[D25N] (green or gray). DRV binding (A) was observed in two orientations with relative occupancies of 55% (red) to 45% (black) in PR·DRV (2IEN (32)) and 76% (green) to 24% (gray) in PR[D25N]·DRV. RPB binding (B) was observed in a single orientation (red in PR (2AOD (37)) or green in PR[D25N]). Distances between the active site residues and the inhibitor (black arrows) are indicated in angstroms. Residues P4-P3′ of the RPB inhibitor are marked, and alternate conformations of P1′ and P3′ side chains are indicated in gray.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 13459-13470) copyright 2008.

Figures were selected by an automated process.