WhiB-like developmental proteins and their iron-sulfur cluster dependent protein-protein interactions

Stewart, Melissa (2021) WhiB-like developmental proteins and their iron-sulfur cluster dependent protein-protein interactions. Doctoral thesis, University of East Anglia.

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WhiB-like (Wbl) proteins are iron-sulfur (Fe-S) cluster proteins unique to Actinobacteria. This work reports the characterisation of developmental Wbl proteins from Streptomyces Venezuelae, WhiB (SvWhiB) and WhiD (SvWhiD), along with biophysical studies of their iron- sulfur clusters and their protein-protein interactions. Like several Wbl proteins previously characterised, both SvWhiB and SvWhiD bind a [4Fe-4S] cluster that is sensitive to O2, and, of the two the [4Fe-4S] SvWhiB is particularly fragile. Both proteins also showed a distinctive high sensitivity to nitric oxide (NO). Kinetic studies revealed that SvWhiD reacts in a concerted manner with ~9 NO per cluster. Characterisation studies also revealed that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. The mechanisms by which Wbl proteins function as transcriptional regulators has yet to be clearly demonstrated. In some cases, at least, this occurs via an interaction with another protein. Both SvWhiB and SvWhiD interact with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB 4). Non-denaturing ESI-MS experiments was used to determine the dissociation constant (Kd) for the SvWhiD-σHrdB 4 complex as ∼0.7 μM, consistent with a relatively tight binding interaction. The complex formation was cluster dependent and a reaction with NO, which was complete at 8-10 NO molecules per cluster, resulted in dissociation of the Wbl: σHrdB4 into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB 4, consistent with protection of the cluster in the complex.

An interaction unique to WhiB is via a (non-Wbl) transcription factor called WhiA. Characterisation studies showed both S. venezuelae and S. coelicolor WhiA bind zinc, and that the zinc-bound form of SvWhiA was responsible for direct interactions with DNA. In vitro EMSA studies confirmed direct binding of SvWhiA to DNA, but also revealed the previously unobserved direct binding of SvWhiB to the promoter regions of genes involved in sporulation initiation.

The additional study of an iron-sulfur cluster transcriptional regulator focused on RirA (Rhizobial iron regulator A), a global iron regulator in Rhizobium. RirA coordinates a [4Fe-4S] cluster via three conserved cysteines and has been shown to sense iron via a labile fourth iron. An N8D RirA variant was generated based on the sequence of the previously structurally characterised NsrR, in which the fourth ligand is an aspartate residue. Strikingly, the N8D variant was found to have lost its iron sensing abilities but exhibited increased cluster stability in the presence of oxygen when compared to wild type RirA.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Chris White
Date Deposited: 22 Sep 2021 08:48
Last Modified: 31 Mar 2023 01:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/81484


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