Background:  Drug resistance occurs when mutations in the target protein allow the protein to retain function while no longer being inhibited efficiently by the drug. In the case of HIV-1 protease, drug resistance occurs when the enzyme retains the ability to cleave the gag and pol polyproteins in at least 9 different locations, which allows viral maturation even in the presence of protease inhibitors. At first inspection development of drug resistance for HIV-1protease would appear to be particularly difficult as all of the currently prescribed protease inhibitors are competitive inhibitors that bind in the center of the active site. Nevertheless many viable drug-resistant mutations occur in viral samples taken from patients due to the infidelity of the reverse transcriptase and the selective pressure of the virus evolving in the presence of protease inhibitors. These drug-resistant mutations effectively render current therapies ineffective.

Methods: Crystal structures of substrate complexes with an inactive (D25N) protease were determined. Diffraction data was collected, processed and refined, using standard crystallographic techniques. The structures are compared graphically with each other and with other crystal structures of inhibitor complexes determined in our and other laboratories.

Results: All of the substrates recognized by HIV protease appear to adopt a particular shape or “substrate envelope” that is not obvious from the non-homologous primary sequences of the substrate sites. This has been determined by superimposing the various crystal structures determined in our laboratory. Many of the drug resistant mutations that occur in HIV protease occur in regions that do not directly contact the substrate envelope, allowing viral processing to occur even in the presence of inhibitors.

Conclusions:  Drug resistance occurs at locations in HIV protease that are least likely to impact substrate recognition.

Keywords: HIV protease; substrate recognition; crystallography