PDBsum entry 2pym

Hydrolase

PDB id: 2pym

Contents

Protein chains

99 a.a. *

Ligands

1UN

Waters ×138

* Residue conservation analysis

PDB id:

2pym

Name:

Hydrolase

Title:

HIV-1 pr mutant in complex with nelfinavir

Structure:

Protease retropepsin. Chain: a, b. Synonym: HIV-1 protease. Engineered: yes. Mutation: yes

Source:

Human immunodeficiency virus 1. Organism_taxid: 11676. Strain: type b. Gene: gag-pol. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.90Å R-factor: 0.195 R-free: 0.244

Authors:

P.Rezacova,M.Kozisek,K.Saskova,J.Brynda,J.Konvalinka

Key ref:

M.Kozísek et al. (2007). Molecular analysis of the HIV-1 resistance development: enzymatic activities, crystal structures, and thermodynamics of nelfinavir-resistant HIV protease mutants. J Mol Biol, 374, 1005-1016. PubMed id: 17977555 DOI: 10.1016/j.jmb.2007.09.083

Date:

16-May-07 Release date: 26-Feb-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 3 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: 10.1016/j.jmb.2007.09.083

J Mol Biol 374:1005-1016 (2007)

PubMed id: 17977555

Molecular analysis of the HIV-1 resistance development: enzymatic activities, crystal structures, and thermodynamics of nelfinavir-resistant HIV protease mutants.

M.Kozísek, J.Bray, P.Rezácová, K.Sasková, J.Brynda, J.Pokorná, F.Mammano, L.Rulísek, J.Konvalinka.

ABSTRACT

Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that cleaves viral polyproteins into mature proteins, thus leading to the formation of infectious particles. Protease inhibitors (PIs) are successful virostatics. However, their efficiency is compromised by antiviral resistance. In the PR sequence of viral variants resistant to the PI nelfinavir, the mutations D30N and L90M appear frequently. However, these two mutations are seldom found together in vivo, suggesting that there are two alternative evolutionary pathways leading to nelfinavir resistance. Here we analyze the proteolytic activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1 PRs harboring the D30N and L90M mutations alone and in combination with other compensatory mutations. Vitality values obtained for recombinant mutant proteases and selected PR inhibitors confirm the crucial role of mutations in positions 30 and 90 for nelfinavir resistance. The combination of the D30N and L90M mutations significantly increases the enzyme vitality in the presence of nelfinavir, without a dramatic decrease in the catalytic efficiency of the recombinant enzyme. Crystal structures, molecular dynamics simulations, and calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M) were used to augment our kinetic data. Calorimetric analysis revealed that the entropic contribution to the mutant PR/nelfinavir interaction is less favorable than the entropic contribution to the binding of nelfinavir by wild-type PR. This finding is supported by the structural data and simulations; nelfinavir binds most strongly to the wild-type protease, which has the lowest number of protein-ligand hydrogen bonds and whose structure exhibits the greatest degree of fluctuation upon inhibitor binding.

Selected figure(s)

Figure 1.
Figure 1. Superimposition of structures of HIV-1 PR mutants in complex with nelfinavir. The positions of mutations are indicated by spheres and numbered. The protease is shown in ribbon representation. The D30N/N88D mutant is colored red, the D30N/A71V mutant yellow, the D30/L90M mutant green and the D30N mutant blue. Nelfinavir is represented as a stick model and its solvent accessible surface is colored according to electrostatic potential: blue, positive; red, negative (calculated with DS ViewerPro 6.0 (Accelerys Software Inc.)).

Figure 3.
Figure 3. Crystal structures of PR mutants in complex with NFV. (a) Two orientations of NFV in the mutant PR active site. The 2F[o]–F[c] electron density map is contoured at the 1.0σ level. The carbon atoms for the two NFV conformations are colored yellow and light red, respectively. Oxygen, nitrogen and sulfur atoms are colored red, blue and gold, respectively. Catalytic aspartate residues are shown with carbon atoms colored gray. (b) Superposition of D30N mutant with wild-type complex structure. Top view of the PR active site. Nelfinavir with residues 30 are shown in stick representation with the carbon atoms are colored gray and light red for the wild-type and D30N mutant, respectively. Oxygen, nitrogen and sulfur atoms are colored red, blue and gold, respectively. (c) Close van der Waals contact of the M90 side-chain with the main-chain of the catalytic residue. Van der Waals surfaces are shown as meshes for catalytic aspartate and residue 90 in the D30N mutant and the D30N/L90M mutant. (d) Hydrogen bond network formed by residue 88 in wild-type and D30/N88D PR. The wild-type structure is represented by gray carbon atoms, while the mutant structure is represented by green carbon atoms. Water molecules (shown as spheres) are colored correspondingly. Hydrogen bonds are represented by dotted lines. The water mediated contact between D88 and N30 in the mutant structure is represented by red lines. (e) Plot showing the RMSDs for the positions of main-chain and side-chain atoms of individual residues after superimposition of the D30N PR and D30N/A71V PR structures.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 374, 1005-1016) copyright 2007.

Figures were selected by the author.