Maintaining Proteome Integrity: Structural and Functional Dynamics of Bacterial Protein Quality Control

Abstract

Cells often encounter a variety of environmental stresses such as heat shock and oxidative stress, which can compromise the stability of biomolecules. To maintain a healthy proteome, protein quality control systems composed of chaperones to assist folding and proteases to degrade misfolded proteins have been evolved. In Gram-negative bacteria like E. coli, protein quality control is specialized due to the presence of two membranes. The periplasmic serine-protease DegP, plays a key role during heat stress by switching between chaperone and protease functions, which is accompanied by large structural rearrangements. In this thesis, the oligomeric assemblies of DegP have been investigated using solution 19F NMR. We show that site-specific 19F labelling of tyrosines in large protein complexes provides significant advantages for probing conformational changes, identifying previously unobserved local asymmetries in the DegP hexamer. The E. coli protein quality control system also heavily relies on ATP-dependent proteins such as the AAA+ protease FtsH. FtsH forms a homohexamer in the inner membrane where it exerts both a regulatory function in selective degradation, and quality control of misfolded membrane proteins. Although FtsH is essential in E. coli, due to its role in maintaining the cell envelope, and its potential as an antibiotic target, important structural and functional aspects remain poorly understood. In this thesis, I have focused on characterizing the dynamical and functional properties of FtsH cytoplasmic domain using solution NMR spectroscopy as main method. Backbone relaxation of the AAA+ domain reveals interplay between its two subunits, while side-chain dynamics reflect the influence of the more rigid protease domain. I further show that a hexameric, active FtsH construct can be used to probe functional aspects of the ATP cycle. This revealed allosteric coupling between the AAA+ domain and the protease domain as well as nucleotide release as potential rate limiting step. Using site-specific 19F labelling of the functionally essential pore loop residue F228, we demonstrate that nucleotide binding stabilizes multiple conformational states. Consistent with a sequential ATP hydrolysis mechanism, hexamer symmetry is broken, resulting in distinct pore loop conformations with unequal populations. Together, this work provides insights into the structural and functional dynamics of two key machineries in the bacterial protein quality control.