5. Discussion
5.1. Structural and biochemical analysis of MAP FurA
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et al. 2012; Ernst et al. 2005; Palyada, Threadgill, and Stintzi 2004). This needs to be addressed in further studies, either by Electrophoretic Mobility Shift Assay (EMSA) experiments or other interaction studies. It is also possible, that in the obtained apo-SAXS structure the structural zinc is also missing, hence inducing a conformational change of MAP FurA and a possible fourth MAP FurA conformation, that is still able to bind the DNA.
Figure 42: Putative gene regulation of MAP FurA under different stress conditions, as indicated by the obtained SAXS results. If enough iron is available, MAP FurA is either inducing or repressing gene expression of specific genes via binding to specific DNA binding motifs. Upon oxidative stress, a conformational change into the oxidized open form takes place, leading to dissociation of the protein from the DNA. Upon iron starvation, the metal ion gets misplaced from the protein, inducing a conformational change into the open apo-form of MAP FurA. This results in the dissociation of the protein from the DNA and gene regulation is hindered. The open forms could then be degraded by a protease until iron is available again or peroxide levels decreased again. Dashed lines indicate putative processes, that were not studied yet. The images were generated with the PyMOL molecular graphics package (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC (Schrödinger, LLC 2015)).
MAP FurA adopts the open conformation when the protein is in its non-oxidized form.
This indicates, that upon oxidative stress, MAP FurA undergoes a conformational change leading to dissociation from the DNA, enabling the stop of the gene regulation and hence adaptation to environmental changes.
121 The overall shape similarity of MAP FurA to Magnetospirillum gryphiswaldense MSR-1 Fur-Mn2+ could indicate a similar regulation mechanism for MAP FurA (as described in chapter 4.1.5). In further studies, the complex formation of MAP FurA to different promoter sequences should be analyzed to study the binding mechanism of MAP FurA DNA binding with regards to the availability of metals or under oxidative stress.
Information from native MS, SAXS or with FPLC experiments could elucidate, if positive and negative regulation may be facilitated by the binding of one or two homodimers of MAP FurA to the cognate promoter sequence. Till now, it has not been possible to detect any DNA binding of MAP FurA with EMSA. This is exacerbated by the fact, that no conclusive consensus sequence for MAP FurA binding could be found by sequence analysis (Eckelt 2014).
CD measurements revealed significant conformational changes of MAP FurA after treatment with different reagents (see chapter 4.1.6.). Firstly, the untreated sample had large amounts of α-helices (~47%) and ~18.6% β-sheets. A drastic decrease of the α-helical portion was observed upon iron addition. For the redox sensing Fur homologs, a shift towards lower α-helical protein portion was detected as well (PDB entries: 4raz, 3f8n (Deng et al. 2015; Jacquamet et al. 2009). However, there was no change observed in the β-sheet content of the proteins. This might indicate a major conformational change for MAP FurA, that differs from PerRBS and Magnetospirillum gryphiswaldense MSR-1 Fur-Mn2+. After H2O2 treatment a further conformational change was observed. This validates the proteins sensitivity towards oxidative stress on a structural level. This further conformational change could lead to the dissociation of the MAP FurA dimer from the DNA, if significantly high amounts of H2O2 are threatening the cell.
It was further shown, that upon peroxide stress a dissociation of PerRBS dimer is induced (Traoré et al., 2006). This is not the case for MAP FurA, as there is no rapid dimer dissociation after treatment with H2O2 (see chapter 4.1.7.). Even after 6 h, where PerRBS
was completely monomeric, only a small fraction of the whole MAP FurA protein was monomeric, whereas most of the protein was dimeric. Moreover, several other peaks were detected, indicating proteolytic processes. It seems, that some regulatory mechanisms between PerRBS and MAP FurA differ, despite their homology and more information about those processes could elucidate the mode of action of MAP FurA in response to host-induced defense mechanisms.
Several Fur homologs are able to bind different metals with different affinities to further broaden the regulation abilities. Therefore, the influence of different metals on MAP FurA were examined (see chapter 4.1.8). Thermofluor analysis revealed, that the protein does not show severe differences relating to the melting temperature Tm. Only the addition of iron increased the Tm slightly, which corresponds to the proposed binding of iron to MAP FurA for it to be in its active state and is maybe needed for the stabilization of the dimer. EDTA addition decreased the stability of MAP FurA. This could be related to the fact, that in PerRBS, the demetallized apo-PerRBS gets degraded by the LonA protease and is hence useless (Ahn and Baker 2016). Binding studies of MAP FurA to zinc, iron and manganese cations revealed that MAP FurA is able to bind all three metals, as revealed by SDS-PAGE (see chapter 4.1.9). This further suggests that MAP FurA not only uses iron as a cofactor, but is also able to bind manganese ions as well. In Bacillus subtilis, the addition of manganese to the growth media led to a repression of the PerRBS mediated gene regulation to oxidative stress, whereas the addition of iron caused the normal expression profile that occurs, if oxidative stress is present (Fuangthong et al. 2002).
Therefore, under physiological cytosolic conditions, manganese binds to the protein and genes are differentially regulated. If oxidative stress occurs, iron levels rise, iron binds to PerRBS and gene expression is altered. The results for MAP FurA indicate, that the regulation might be similar in MAP. It was shown, to my knowledge for the first time, that a mycobacterial Fur protein is able to bind manganese. So far it could only be shown for FurA from Mycobacterium tuberculosis that it can bind zinc and iron (Lucarelli 2006).
Interestingly, the unoxidized portion of MAP FurA was able to bind to zinc ions. This might indicate, that the essential structural zinc ions, like they are present in PerRBS, are not bound to MAP FurA when treated with metal chelators. An apo-form lacking any metal ion has previously been reported (Deng et al. 2015). It must be noted, that Fur proteins routinely show two bands when resolved via SDS PAGE (Lee and Helmann 2006). It was shown for PerRBS, that the lower migrating band contains the structural important zinc ion, whereas the higher migrating band does not. This effect was enhanced if the protein was pretreated with EDTA or high levels of H2O2 (Lee and Helmann 2006).
To further investigate the structural changes more thoroughly, data at atomic resolution would be beneficial. Crystallization trials led to some promising conditions, nonetheless obtaining crystals suitable for X-ray diffraction analysis failed (see chapter 4.1.3).
123 Interestingly, the condition that produced microcrystals (Figure 8C) was only slightly modified from the crystallization of the MAP FurA homolog from Magnetospirillum gryphiswaldense (Liu, Chen, and Wu 2012). Unfortunately, optimization of crystals failed so far. Further optimization of the protein quality, e.g. the addition of additives or a further purification step could be beneficial. Furthermore, the oxidation status of the protein could be a problem. Most of the homologs are metal uptake regulators and not redox sensing proteins and hence the crystallized version is oxidized, which does not seem problematic for crystallization success in those cases. Citric acid is a metal chelator, resulting in the formation of apo-FurA or a mixture of oxidized and unoxidized protein.
The purification under anaerobic conditions could be tried to circumvent the mixture of protein species over time. Furthermore, the slow transition from MAP FurA dimer to monomer upon metal deprivation could lead to inhomogeneities, hindering high quality crystal formation.