PDBsum entry 2nxm

Hydrolase/hydrolase substrate

PDB id: 2nxm

Contents

Protein chains

99 a.a. *

Ligands

ALA-GLN-THR-PHE-TYR-VAL-ASP-GLY

Waters ×71

* Residue conservation analysis

PDB id:

2nxm

Name:

Hydrolase/hydrolase substrate

Title:

Structure of HIV-1 protease d25n complexed with the rt-rh analogue peptide gly-ala-gln-thr-phe Tyr-val-asp-gly-ala

Structure:

Protease retropepsin. Chain: a, b. Synonym: HIV-1 protease. Engineered: yes. Mutation: yes. Analogue of rt-rh pol protease substrate peptide. Chain: p. Fragment: decapeptide fragment. Engineered: yes.

Source:

HIV-1 m:b_arv2/sf2. Organism_taxid: 11685. Strain: sf2. Gene: pol. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: this sequence was custom-designed and then purchased commercially

Resolution:

2.25Å R-factor: 0.190 R-free: 0.244

Authors:

M.Prabu-Jeyabalan,E.Nalivaika,C.A.Schiffer

Key ref:

M.D.Altman et al. (2008). Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease. Proteins, 70, 678-694. PubMed id: 17729291 DOI: 10.1002/prot.21514

Date:

17-Nov-06 Release date: 18-Sep-07

Protein chains

P03369  (POL_HV1A2) -  Gag-Pol polyprotein from Human immunodeficiency virus type 1 group M subtype B (isolate ARV2/SF2)

Seq: 1437 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.- - ?????
• Enzyme class 2: E.C.2.7.7.49 - RNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 3: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 4: E.C.3.1.-.-
• Enzyme class 5: 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 6: E.C.3.1.26.13 - retroviral ribonuclease H.
• Enzyme class 7: 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: 10.1002/prot.21514

Proteins 70:678-694 (2008)

PubMed id: 17729291

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease.

M.D.Altman, E.A.Nalivaika, M.Prabu-Jeyabalan, C.A.Schiffer, B.Tidor.

ABSTRACT

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods-namely, charge optimization and protein design-were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT-RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a 10-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease.

Selected figure(s)

Figure 1.
Figure 1. Molecular environments of the Glu-P3 (A) and Thr-P2 (B) peptide residues found to be suboptimal for electrostatic binding in the RT-RH crystal complex. The position of the P3 glutamate (A, atom colors, center) is pointed away from Arg8B and makes contact with Phe53A. Calculations suggest an alternative conformation (yellow) that makes better electrostatic interactions with Arg8B at the expense of packing. The wild-type P2 threonine residue (B, center) is situated in a pocket composed of four hydrophobic residues and makes no polar interactions. The crystal structure of a designed mutant peptide with a single threonine-to-valine substitution at the P2 position exhibits a structural rearrangement of the Glu-P3 residue (C, atom colors) as compared to the starting sequence (A). This rearrangement is well supported by calculation (A, C, yellow).

Figure 4.
Figure 4. Superposition of the crystal structures for the RT-RT peptide (green), Peptide 1 (cyan), Peptide 2 (purple), and Peptide 3 (yellow) exhibits structural similarity.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 70, 678-694) copyright 2008.

Figures were selected by an automated process.