The Alphaproteobacterial General Stress Response: Investigating Protein Structure, Dynamics, and Regulatory Mechanisms

Author

Danielle Swingle, The Graduate Center, City University of New YorkFollow

Date of Degree

2-2025

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Kevin H. Gardner

Committee Members

Sean Crosson

Derrick Brazill

Ruth Stark

Daniel Keedy

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Keywords

stress response, two-component systems, histidine kinase, blue light sensor, response regulator

Abstract

Bacteria are relatively simple organisms that face a wide variety of environmental challenges. Their survival is determined by their ability to sense and respond to changes in their surroundings. Alphaproteobacteria commonly accomplish this using the general stress response (GSR). During the first step in this system, a signal is detected by a sensor histidine kinase (HK), modulating autophosphorylation within its kinase domain. HKs involved in the GSR generally belong to the HWE/HisKA2 family, a lesser studied group of HKs with noncanonical features. The signal is propagated when the HK phosphorylates the next protein in the pathway, PhyR. Once activated, PhyR acts as an anti-anti-s factor, binding the anti-s factor NepR. This releases the sigma factor sigma^EcfG, which is normally inhibited by NepR in the absence of a stress signal. Once sigma^EcfG is free, it can activate transcription of the stress response genes. Many aspects of this system and its key players have yet to be elucidated. Some of these are addressed in this thesis, which is an investigation into the structure, dynamics, and regulatory mechanisms of a set of Alphaproteobacterial GSR proteins. It is broken down into two parts.

Though oligomeric state is typically conserved along with function across evolution, exceptions such as the hemoglobins exemplify how altered oligomerization can enable new regulatory mechanisms. In our first study, we examine the conservation of oligomeric state in HKs, a large class of widely distributed prokaryotic environmental sensors. While most studied HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture. Our prior discovery of the monomeric soluble EL346, a photosensing Light-Oxygen-Voltage (LOV)-HK, is one example. To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim (PAS)-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.

In our second study, we focus on the protein RT-HK, another LOV-HK from the HWE/HisKA2 family that was identified in the first study. This protein has a monomer/dimer equilibrium tunable by light signal and nucleotide binding, and uses an inverted signaling logic. Here, we further investigate these atypical behaviors of RT-HK and characterize the downstream signaling network. Using hydrogen-deuterium exchange mass spectrometry, we find that despite its inverted logic, RT-HK uses a signal transduction mechanism similar to typical light-activated systems. Mutagenesis reveals that the protein autophosphorylates in trans, and the length of its Ja helix affects net autophosphorylation levels. To explore the downstream effects of RT-HK, we identified two GSR genetic regions, each encoding a copy of the central regulator PhyR. In vitro phosphotransfer measurements revealed that RT-HK signals only to one, and at an increased intensity in the dark, suggesting transfer of its reversed logic. Solving the X-ray crystal structures of both PhyRs revealed a substantial shift in the receiver domain of one paralog. We probed further down the GSR pathway using NMR to determine that a NepR homolog interacts with both unphosphorylated PhyRs, an interaction that is decoupled from activation in one paralog. This work offers a more comprehensive understanding of HWE/HisKA2 family signal transduction in paralogous GSR networks.

Recommended Citation

Swingle, Danielle, "The Alphaproteobacterial General Stress Response: Investigating Protein Structure, Dynamics, and Regulatory Mechanisms" (2025). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/6072