Structural and functional studies on DNA damage inducible protein 1 (Ddi1) from protozoa

Abstract

Structural and functional investigations on DNA-damage inducible protein1 (Ddi1) from Entamoeba histolytica, Trypanosoma cruzi and Toxoplasma gondii have been carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs reduced the infection caused by many parasitic protozoa such as Trypanosoma and Leishmania species that are responsible for prevalent opportunistic diseases in AIDS patients. The retroviral protease-like domain (RVP) present in Ddi1 proteins of these organisms was identified as the target of these drugs. The binding of the RVP domain of Ddi1 from E. histolytica, T. cruzi and T. gondii with HIV protease inhibitors; and the binding of ubiquitin and K48-linked diubiquitin with the ubiquitin associated domain (UBA) have been established by Biolayer Interferometry (BLI). The crystal structure of the RVP domain of Ddi1 from T. gondii (ToxoDdi1-RVP) shows that it forms a homodimer similar to that observed in HIV protease and the reported structures of the same domain from S. cerevisiae, L. major and human. The ‘flap’ regions in ToxoDdi1-RVP are similar to the flaps of HIV protease which close-in upon substrate/inhibitor binding. Both the ‘flap’ regions are clearly visible in the electron density maps. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. Comparison of the structure of ToxoDdi1-RVP with the available crystal structures of Ddi1-RVP from other organisms revealed that the active site architecture is conserved in all the proteins with differences in the back β-sheet topology and the size of the binding cavity.