Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Ronen Q. Marmorstein

Abstract

Amino-terminal acetylation is a co-translational process that occurs on a majority of all eukaryotic proteins. This modification mediates a wide range of biological processes including but not limited to cellular apoptosis, enzyme regulation, protein localization and the N-end rule for protein degradation. The amino-terminal acetyltransferase (NAT) enzymes that catalyze this reaction are distinguished from one another on the basis of substrate specificity, which is dictated by the identity of the amino-terminal sequence of the substrate protein. The NatA complex harbors the greatest diversity for substrate selection and is responsible for acetylating a majority of all proteins that undergo this modification in vivo. Interestingly, the major NATs exist as obligate heterodimers with one unique catalytic subunit and an additional unique auxiliary subunit that both activates the catalytic component and anchors the complex to the ribosome. Aberrant expression of the NatA catalytic subunit, Naa10p, as well as the auxiliary subunit, Naa15p, has been found in numerous cancer cell tissues; consequently, NatA is an emerging target for chemotherapeutic development.

Despite the ubiquitous nature of this modification as well as its role in oncogenesis, there is no structural data available for any eukaryotic NAT protein. Inspired by this, we determined the x-ray crystal structures of a series of NAT enzymes, highlighted by the structure of the 100 kDa NatA complex in the absence and presence of a bisubstrate peptide-CoA conjugate inhibitor as well as the structure of the uncomplexed Naa10p catalytic subunit. Additionally, we determined the structure of the independently active NatE bound to CoA and a substrate peptide fragment and the structure of an archaeal NAT enzyme that acetylates NatA and NatE substrates. These structures and accompanying biochemical assays are used to delineate mechanisms of substrate binding specificity and catalysis employed by this family of enzymes. This work provides the first scaffold for the design of NAT-specific inhibitors and lays a foundation for understanding NAT activity at the molecular level. Furthermore, this study could have implications for mechanisms by which regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

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