Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Sessa, William

Abstract

Cis-prenyltransferses (cis-PTases) constitute a family of enzymes involved in the synthesis of isoprenoid lipids required for various biological functions across all domains of life. The eukaryotic cis-PTase catalyzes the rate-limiting step in the synthesis of dolichyl phosphate, an indispensable glycosyl carrier lipid required for protein glycosylation in the lumen of endoplasmic reticulum. Based on enzyme composition, cis-PTases can be either homomeric or heteromeric enzymes. The human cis-PTase possesses a heteromeric configuration consisting of the two evolutionary related subunits: NgBR (dehydrodolichyl diphosphate synthase accessory subunit, first identified as a Nogo-B receptor) and DHDDS (dehydrodolichyl diphosphate synthase catalytic subunit). Recently, several mutations in both subunits have been reported to associate with various human diseases, collectively known as congenital disorders of glycosylation (CDG), including severe CDG type I, developmental and epileptic encephalopathy, and autosomal recessive retinitis pigmentosa. In addition, mutations on the NgBR subunit have been recently reported in patients suffering from early onset of Parkinson’s disease (EOPD). Despite its crucial role in the protein glycosylation process, the molecular mechanism of heteromeric cis-PTases remains poorly understood due to lack of structural-functional studies on these enzymes, in contrast to homodimeric cis-PTases which have been extensively studied. Therefore, in this dissertation, I illustrate the first crystal structure of a heteromeric, human cis-PTase NgBR/DHDDS complex solved at 2.3 Å. The structure revealed novel features that were not previously observed in homodimeric enzymes, including a new dimeric interface formed by a unique C-terminus in DHDDS and a novel N-terminal segment in DHDDS serving as a membrane sensor for lipid activation. In addition, the structure elucidated the molecular details associated with substrate binding, catalysis, and disease-causing mutations. Finally, the structure provided novel insights into the mechanism of product chain elongation, an interesting yet one of the most enigmatic topics on prenyl chain elongating enzymes. In summary, the crystal structure advances our understanding of the molecular mechanism of heteromeric cis-PTase enzymes.

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