All ETDs from UAB

Advisory Committee Chair

Bingdong Sha

Advisory Committee Members

Chenbei Chang

James Collawn

Sthanam Vl Narayana

Peter Edward Prevelige Jr

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The unfolded protein response is one mechanism utilized by endoplasmic reticulum (ER) to maintain the homeostasis between ER protein folding machinery and ER proteins. UPR is induced by three ER transmembrane sensors: IRE1, ATF6 and PERK. PKR-like ER kinase (PERK) can sense the ER stress signal through its luminal domain to activate its cytoplasmic kinase domain catalytic activity. PERK kinase domain belongs to the eIF2α family. Members in this family are activated after being autophosphorylated at their activation loops and then specifically phosphorylate eIF2α at its Ser51 position. Phosphorylation of eIF2α can shutdown the cytosol protein translation from the initiation stage. Studies on PERK luminal domain and kinase domain indicated that PERK may form un-clustered homo-dimer in its inactive form. The crystal structure of PERK kinase domain determined in this dissertation revealed an intermediate conformation between inactive conformation and active conformation. In this conformation, the activation loop in PERK kinase domain C-lobe has been phosphorylated at a conserved Thr residue, T980, and stabilized by this phosphorylation. In addition, helix 8 in PERK KD C-lobe is fixed through the interactions with stabilized activation loop and ready for substrate bindings. A "line-Up" model for PERK KD autophosphorylation was proposed in this dissertation base on the dimer orientation in this structure. This can be also used to explain mechanism of PERK pathway activation. The translocation of mitochondria matrix proteins is conducted by an IMM translocase TIM23. Tim44 is an essential, peripheral membrane protein in TIM23 translocation pathway, which associates with IMM through its C-terminal domain and recruits the import motor to the TIM23 channel. It can be localized either on the membrane or in the matrix. X-ray Crystallographic and biochemical studies on Tim44 CTD in this dissertation revealed that the N-terminal helices A1 and A2 in Tim44 CTD are amphipathic and critical for Tim44 membrane binding. Hydrophobic residues in A1 and A2 contribute to the hydrophobic interactions between Tim44 and IMM. Base on these data, a membrane-induced conformational change model has been proposed to explain the mechanism for Tim 44 membrane binding.

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