4EP2

Crystal Structure of inactive single chain wild-type HIV-1 Protease in Complex with the substrate RT-RH

Summary for 4EP2

• Entry DOI: 10.2210/pdb4ep2/pdb
• Related: 4EP3 4EPJ 4EQ0 4EQJ
• Descriptor: protease, tethered dimer, substrate RT-RH, GLYCEROL, ... (5 entities in total)
• Functional Keywords: hiv-1 protease, specificity design, drug design, protease inhibitors, aids, aspartyl protease, hydrolase, hydrolase-hydrolase substrate complex, hydrolase/hydrolase substrate
• Biological source: HIV-1 M:B_ARV2/SF2 (HIV-1); More
• Cellular location: Gag-Pol polyprotein: Host cell membrane; Lipid-anchor. Matrix protein p17: Virion membrane; Lipid- anchor . Capsid protein p24: Virion . Nucleocapsid protein p7: Virion . Reverse transcriptase/ribonuclease H: Virion . Integrase: Virion : P03369
• Total number of polymer chains: 2
• Total formula weight: 23093.00

Authors

Schiffer, C.A.,Mittal, S. (deposition date: 2012-04-16, release date: 2012-06-06, Last modification date: 2024-02-28)

Primary citation

Alvizo, O.,Mittal, S.,Mayo, S.L.,Schiffer, C.A.
Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.
Protein Sci., 21:1029-1041, 2012

PubMed Abstract: HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

PubMed: 22549928
DOI: 10.1002/pro.2086

Experimental method

X-RAY DIFFRACTION (1.9 Å)