HIV-1 protease tethered heterodimer-pepstatin-a complex: NMR characterization

Abstract

Wild type HIV-1 protease is a homodimer that plays a crucial role in the function of the virus and hence has been a target of anti HIV (human immunodeficiency virus) drug design. However, heterodimers generated by different mutations in the individual monomers have been seen to have different characteristics and thus have been used as macromolecular inhibitors of enzyme activity. Further, during the course of clinical treatment, it is conceivable that more than one mutant species exist inside a cell, resulting in the production of both homo- and heterodimers of the enzyme. In this context we have investigated here by NMR, the characteristic features of a particular heterodimer complexed to the peptide inhibitor pepstatin-A. The heterodimer has a GGSSG linker joining the two monomers head to tail and one of the monomers has a C95M mutation that lies in the dimerization domain. NMR backbone assignments have revealed that there is an asymmetry between the two monomer units. The secondary structural characteristics of the protein in the complex are almost identical to those in the crystal structure of the wild type homodimer protein complexed to the inhibitor, acetyl pepstatin. Thus neither the covalent linker nor the mutation seems to affect the gross solution structural features of the protease. Some local differences are, however, seen near the site of mutation. The inhibitor sitting in the active site cavity exhibits a flip-flop motion. Amide proton deuterium exchange studies reveal different local stabilities of the individual residues and also differences between the two monomer units. To our knowledge, this is the first NMR report characterizing a heterodimer of HIV-1 protease and forms a basis for future detailed investigation with different heterodimers, inhibitors, etc.