Structural Biochemistry and Inhibition of CaaX Protein Prenyltransferases From Human Pathogens

Abstract

Protein prenylation is a post-translational lipid modification required for proper function by over 100 proteins in the eukaryotic cell. Proteins that receive this modification mediate a wide variety of functions in the cell, including critical signal transduction events. A family of structurally-related protein prenyltransferase enzymes carry out this reaction: protein farnesyltransferase (FTase), protein geranylgeranyltransferase-I (GGTase-I) and Rab geranylgeranyltransferase (GGTase-II or Rab GGTase). The focus of this dissertation will be on CaaX protein prenyltransferases, FTase and GGTase-I, which recognize a defined C-terminal motif on substrate proteins: cysteine (C), followed by two generally aliphatic amino acids (aa) and a variable (X) residue. Protein farnesyltransferase (FTase) catalyzes the addition of a 15-carbon isoprenoid lipid to certain CaaX proteins, while protein geranylgeranyltransferase-I catalyzes the addition of a 20-carbon lipid. FTase and GGTase-I have been shown to be important drug targets in the fight against cancer, as many of the prenylated signal transduction proteins play significant roles in oncogenesis. More recently, protein prenyltransferases have been identified in human pathogens, and these orthologs also show promise as drug targets for treating infectious diseases. The research in this dissertation seeks to understand the structural biochemistry and mechanisms inhibition of protein prenyltransferase orthologs from human pathogens. Molecular cloning techniques, biochemical assays, and macromolecular X-ray crystallography are employed to express recombinant proteins and study their structure and function. In this work I present the first X-ray structures of non-mammalian protein prenyltransferases, including the FTases from Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans ; as well as the GGTase-I from Candida albicans. These structures reveal regions of the active sites that diverge sufficiently from mammalian orthologs that selective inhibitors to treat infectious diseases may be developed. In addition, I present the crystal structures of a novel series of FTase inhibitors bound to both mammalian FTase and C. neoformans FTase. The structures of these ethylenediamine-scaffold inhibitors reveal dominant determinants of inhibitor binding, as well as ways that the inhibitors could be modified to bind the FTases from multiple human pathogens. Taken together, the data presented in this dissertation advance our understanding of the structural biology of protein prenyltransferases across multiple species, and these data can be exploited to develop novel treatments for infectious diseases.