Structure-Function Studies On Novel Signaling Regulators

Abstract

This dissertation studies the structures and functions of novel signaling regulators, with Chapter 2-4 focusing on sirtuins and Chapter 5 on the DOCK180 family of Rho GEFs. Sirtuins are NAD-dependent deacetylases that regulate important biological processes. Mammals have seven sirtuins, SIRT1-7. SIRT4-7 have undetectable or weak deacetylase activity. In Chapter 2 we identified SIRT5 as an efficient protein lysine desuccinylase and demalonylase. The preference for succinyl and malonyl groups is accomplished by their interactions with residue Tyr102 and Arg105 of SIRT5. Lysine malonylation and succinylation were identified in mammalian proteins. Additionally, SIRT5 can reverse succinylation in vivo. In Chapter 3 I delineated the desuccinylation reaction of SIRT5 in crystals, including the complex structure of SIRT5 with a bicyclic intermediate. The SIRT5 complex structures will provide insights to the design of SIRT5-specific inhibitors to investigate its biological functions. In Chapter 4, we investigated a sirtuin homologue from the malaria parasite Plasmodium falciparum, PfSir2A, which regulates the expression of surface antigens to evade the detection by host immune surveillance. We present enzymology and structural evidence supporting that PfSir2A preferentially hydrolyzes medium and long chain fatty acyl groups from lysine residues. This would facilitate the development of PfSir2A inhibitors as potential drugs in malaria treatment. As the Guanine nucleotide Exchange Factors (GEFs) of Rho GTPases, the DOCK180 family proteins are key regulators of cell motility, phagocytosis, and adhesion. Mammals have 11 members, DOCK1-11, which are classified into four subfamilies, A through D. The DOCK-C subfamily that includes DOCK6-8 has been proposed to activate both Cdc42 and Rac1. In Chapter 5 I show that DOCK7 promotes very weak activation of non-prenylated Cdc42 or Rac1 in solution, but robust activation of prenylated Cdc42 and Rac1 on model liposomes, demonstrating that the prenylation and membrane localization of GTPases are essential for the activation by DOCK7. Additionally, DOCK7 harbors residues that impart GTPase specificity and these can be mutated to shift a given DOCK7 activity profile. Finally, the DOCK7 possesses a distal site that binds preferentially to the active forms of Cdc42 and Rac1 and thereby forms a possible positive feedback loop in activating GTPases