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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

Year Degree Awarded

2016

Month Degree Awarded

May

First Advisor

Dr Peter Chien

Subject Categories

Bacteriology | Biochemistry | Molecular Biology

Abstract

Intracellular protein destruction is a carefully coordinated and timed regulatory mechanism that cells utilize to modulate growth, adaptation to environmental cues, and survival. In Caulobacter crescentus, a bacterium known for studies of bacterial cell division cycle, the response regulator CpdR couples phosphorylation events with the AAA+ protease ClpXP to provide punctuated degradation of crucial substrates involved in cell cycle regulation. CpdR functions like an adaptor to alter substrate choice by ClpXP, however it remains unclear how CpdR influences its multiple targets. In this thesis, we show that, unlike canonical ClpXP adaptors, CpdR alone does not strongly bind its substrate. Instead, CpdR binds the N-terminal domain of ClpX and prepares (primes) the unfoldase for substrate engagement. This priming creates a recruitment interface that docks multiple substrates and additional adaptor components. Interestingly, adaptor-dependent priming of ClpX avoids concentration-dependent inhibition that limits traditional, scaffolding adaptors. Control of proteolysis during the Caulobacter cell cycle occurs through proteins involved in a complex phosphosignaling network. We show that this phosphosignal disrupts the interaction between CpdR and ClpX. This regulatory mechanism is efficient because phosphorylated CpdR that cannot bind ClpX should, theoretically, free ClpXP for interaction with its other substrates and adaptors. When dephosphorylated, CpdR binds ClpX, allowing ClpXP to temporarily gain capability to target a completely different range of proteins. Mutations in the predicted response regulator output face of CpdR yielded CpdR variants that impact ClpX binding to cause ranked-order change in adaptor activity and biological function. Through a suppressor mutant screen of a CpdR variant that is hugely defective in binding ClpX, we discover a non-phosphorylatable activated variant of CpdR. Our data suggest that this CpdR variant may have adopted a Mg2+-free conformation, giving us insight to the dynamic states that CpdR can adopt during its regulatory function. Together, these results reveal how a single adaptor can command global changes in proteome composition through priming of a protease.

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