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STRUCTURAL BASIS FOR THE FUNCTION AND SUBSTRATE SPECIFICITY OF MODULAR PROTEIN COMPLEXES

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2025-09-05
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Abstract

Assembly of proteins into complexes allows for regulation and diversification of function and is essential for many biological processes. This dissertation describes my work on the function and substrate specificity of different modular protein complexes involved in membrane trafficking and polyketide antibiotic production.Rab1 and Rab11 are essential GTPases that act as gatekeepers of membrane trafficking at the Golgi complex. The two related TRAPP complexes, TRAPPII and TRAPPIII, share the same catalytic subunits, yet function as specific activators of two different GTPases, Rab11 and Rab1, respectively. A steric gating mechanism has been proposed to explain the substrate specificity of TRAPPII but the structural basis for this mechanism remained unknown. I determined cryoEM structures of yeast TRAPPII bound to its substrate Rab11. The structures revealed specific interactions between Rab11 and the TRAPPII complex, how TRAPPII interacts with the membrane, and the mechanistic basis of steric gating. Furthermore, I found that the TRAPPIII complex selects against Rab11 based on repulsive interactions with the catalytic subunits. I also observed an alternative conformation of the TRAPPII complex which may facilitate access of Rab11 to the TRAPP catalytic site. Polyketide synthases (PKSs) are multienzyme complexes that produce polyketides, a diverse class of bioactive natural products. Modular PKSs function like an assembly line, where the catalytic domains are arranged over one or more modules. How multiple domains within a modular PKS rearrange to form specific protein-protein interactions and facilitate sequential catalytic reactions was unknown. I used X-ray crystallography and cryoEM to visualize an intact modular PKS, Lsd14, trapped in two different catalytic stages of the PKS reaction cycle. These structures revealed an asymmetric architecture of dimeric Lsd14 that results in an unequal distribution of the catalytic domains over two reaction chambers. Based on this arrangement, I proposed a pendulum mechanism for the PKS reaction cycle in which the ketoreductase-ACP domains swing back and forth between the two reaction chambers to perform two parallel, but asynchronous chain extension cycles. Taken together, my studies on the TRAPP complexes and PKS revealed new mechanisms explaining how substrate specificity and protein-protein interactions are regulated in the context of protein complexes.

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289 pages

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2023-08

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Keywords

cryoelectron microscopy; Drug discovery; Golgi Apparatus; Membrane Trafficking; Polyketide synthase; Protein structure

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Union Local

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Committee Chair

Fromme, Joseph

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Committee Member

Emr, Scott
Crane, Brian

Degree Discipline

Biochemistry, Molecular and Cell Biology

Degree Name

Ph. D., Biochemistry, Molecular and Cell Biology

Degree Level

Doctor of Philosophy

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Government Document

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Attribution 4.0 International

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dissertation or thesis

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