Investigating the Dynamic Interplay of the MzrA-EnvZ Porin Regulatory Complex: Structural and Functional Characterisation of MzrA

Student thesis: Doctoral Thesis

Abstract

Abstract
Two-component systems are signal transduction pathways that allow bacteria to sense, respond and adapt to changes in their surroundings. They are canonically composed of a membrane-bound histidine kinase (HK) and a cognate response regulator (RR). Upon stimulation, the HK relays the signal to the RR through the transfer of a phosphate group to a conserved aspartate (Asp) residue on the RR. The phosphorylated RR (RR-P) then modulates gene expression in response to the initial stimulus. The EnvZ/OmpR TCS specifically regulates the expression of porins on the outer membrane of E. coli. Bacterial porins, which are associated with nutrient uptake, waste efflux, structural integrity and outer membrane (OM) biogenesis have been linked to antibiotic resistance. This EnvZ/OmpR TCS is activated in response to environmental osmolarity. However, previous studies have shown that MzrA is involved in the activation of this system through its interactions with EnvZ. Despite what is known about this protein, its structure has not been fully explored and its mechanism of EnvZ activation remains largely unclear.
The foundation of the experimental approach was the creation of a single-cysteine library within the periplasmic domain of MzrA, which was previously shown to be important for MzrA-mediated changes in EnvZ signal output. Disulphide crosslinking studies were then carried out to identify what positions within the periplasm were within close proximity. Furthermore, the impact of these MzrA residue substitutions on the EnvZ/OmpR TCS output was investigated through fluorometric analysis.
A protein interaction profile was obtained, which revealed the regions that promote MzrA homodimerisation. The variations in this interaction profile were evaluated in response to the presence or absence of EnvZ. Our data suggests a diverse role played by EnvZ in MzrA- MzrA interactions as EnvZ was seen to stabilise MzrA interactions at specific regions (which include the loop between residues V43 and D51 and the helix formed by residues T79 to A96) while causing the residues at other regions to move farther apart from each other, consequently destabilising interactions at these regions.
Residues D51, K67 and D74 were identified as part of the key residues for the MzrA/EnvZ porin complex. Five residues (I40, D51, G52, F53 and F80) were identified to be essential for the role of MzrA as a modulator, as their alteration led to a reduction in MzrA- stimulated EnvZ activity, bringing it to a level comparable to when MzrA was not present.
Finally, a hydrophobic core within MzrA (made up of residues L38, I40, I55, L59, I78, A89, L93 and L97) was essential for its EnvZ-stimulating activity, as substitutions of these residues reduced EnvZ activity. Molecular dynamics (MD) simulations were employed to investigate the impact of the cysteine mutations on the stability of this hydrophobic core. The analysis revealed fluctuations in all three helices within the periplasmic domain of MzrA. Notably, mutations of the hydrophobic core residues induced a more pronounced increase in the C- terminal helix compared to the other two helices, as revealed by the MD analysis. The C- terminal helix exhibited fluctuations of up to 2.0 Å in the wild-type (WT), escalating to 7.0 Å upon mutation of the hydrophobic core residues. In contrast, helices 2 and 3 exhibited peak fluctuations of approximately 0.5 Å and 0.8 Å, respectively, in the WT. These fluctuations increased to 1.2 Å and 1.0 Å, respectively, when the core residues were substituted.
Our data suggests three regions within the MzrA structure that are essential for MzrA- EnvZ interactions, two of which may be vital for the role of MzrA as a modulator the EnvZ/OmpR TCS. Although this study offers extensive structural and functional insights into MzrA with regards to the EnvZ/OmpR two-component system (TCS), additional investigations on MzrA and EnvZ interactions are imperative for a thorough characterisation of the MzrA- EnvZ porin regulatory complex.
Date of Award17 May 2024
Original languageEnglish
Awarding Institution
  • University of Portsmouth
SupervisorRoger Draheim (Supervisor) & Anthony Lewis (Supervisor)

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