7.1.1 :Purification and characterization of three siderophore-binding receptor proteins ofStreptomyces coelicolorA3(2)
The main aims of this study were to overproduce and purify the three siderophore- binding receptor proteins DesE, CchF and CdtB ofStreptomyces coelicolor A3(2) in
Escherichia coli, and to assess their ability to bind to cognate ferri-siderophores by
monitoring the decrease of the fluorescence of the proteins upon ligand binding.
Initially, the full-length DesE, CchF and CdtB proteins were overproduced inE. coli
with a hexahistidine tag appended to their native N-termini. All three proteins were overproduced in a soluble form, and two were purified by immobilized metal-affinity chromatography (IMAC). CchF, whose His-tag was most probably cleaved by the TAT translocase machinery of E. coli, could not be purified by this method. The other proteins, DesE and CdtB, dimerized very strongly, as indicated by denaturing SDS-PAGE and size-exclusion chromatography (gel filtration).
New plasmids were constructed for the expression of 5’-truncated desE, cchF and
cdtB genes in E. coli. These encoded the recombinant proteins DesE, CchF and
CdtB without the hydrophobic N-terminal signal sequences and lipidation sites. All three proteins were overproduced in soluble form and purified by Ni-IMAC. DesE and CdtB were monomeric, whereas CchF was found to dimerize (using gel filtration). This CchF dimer was dissociated by incubation at 37 °C. The N-terminal His-tags were removed from the proteins using tobacco etch virus (TEV) protease. The identity of the purified proteins was confirmed by peptide mass fingerprinting and ESI-MS analysis.
Circular dichroism analysis of the three proteins showed that two of them, DesE and CchF, were fully folded, whilst CdtB was up to 25% unfolded. This may explain the larger apparent molecular weight (corresponding to a larger hydrodynamic radius) of this protein indicated by gel filtration than the more massive proteins DesE and CchF.
7.1.2 :Intrinsic fluorescence quenching of proteins by ferri-siderophore binding
Solutions of ferri-siderophores were prepared and their concentrations were determined by inductively coupled plasma optical emission spectroscopy (ICP- OES). Ferri-coelichelin was purified from iron-deficient cultures fromStreptomyces
coelicolor using reverse-phase HPLC, utilising a modification of the method of
Lautru et al52, to produce gallium-coelichelin, whose purity was examined by 1H
NMR spectroscopy. Ferri-coelichelin was made by adding an excess of FeCl3 to
gallium-coelichelin.
Solutions of the proteins DesE, CchF and CdtB showed significant decreases in their intrinsic fluorescence upon addition of low concentrations of certain ferric
siderophores. Dissociation constants were calculated from the fluorescence
quenching data by fitting to a one-site binding curve using the equations of Ahnstrom et al85in the software package ORIGIN86.
DesE was found to bind the cognate siderophores ferrioxamine B and ferrioxamine E with dissociation constants in the nanomolar range, but did not bind ferri-coelichelin
with nanomolar affinity. DesE also bound the hydroxamate xenosiderophore
ferrichrome with nanomolar affinity, but ferri-albomycin bound more weakly. CchF bound ferri-coelichelin only, whilst CdtB was found to bind ferrioxamine B and ferri-coelichelin with nanomolar affinity. None of the proteins studied bound theE. coli siderophore ferri-enterobactin, nor did the desferri-siderophores enterobactin, desferri-ferrichrome, and desferrioxamines E and B.
When CdtB was left to stand at room temperature in solution, the fluorescence decreased steadily, likely to be due to its instability. Despite this, the dissociation constants of ferrioxamine B and ferri-coelichelin from CdtB were in the nanomolar range even when it was assumed that only 75% of the protein was folded.
7.1.3 :X-ray crystal structures of DesE and DesE-ferrioxamine B
The X-ray crystal structures of apo-DesE and DesE-ferrioxamine B were determined by our collaborators, at the University of St. Andrews. The co-complex crystal structure confirmed the specific nature of the binding of ferrioxamine B to DesE, as suggested by the fluorescence quenching data. Also, little conformational change was observed in DesE on substrate binding, which was also seen in other siderophore binding proteins.
Analysis of the DesE-ferrioxamine B structure found several aromatic residues around the ferri-siderophore binding site, explaining the large quenching of the
intrinsic fluorescence of DesE upon ferrioxamine B binding. An important
hydrogen-bonding interaction of an arginine residue with the hydroxamate groups
surrounding the ferric iron centre of ferrioxamine B was identified. Also,
ferrioxamine B adopted an unusual configuration never observed before in X-ray crystal structures of this ferri-siderophore.
7.1.4 :Construction of a molecular model of ferric-tris-hydroxamates
Another aim of the project was to construct a molecular model for ferric-tris- hydroxamate complexes (including ferric-tris-hydroxamate siderophores) using the molecular mechanics program DOMMIMOE. This involved parameterization of the program such that it reproduced the geometries observed in X-ray crystallographic structures of ferric-tris-hydroxamates, with energetic information from quantum mechanical calculations on simple ferric-tris-hydroxamates to incorporate the trigonal twist, the main distortion of these complexes seen in the crystal structures.
A suitable parameterization was found by applying the techniques simulated annealing and Nelder-Mead simplex optimization to a cost function whose value was minimal if the geometries and energies produced by the program were close to the desired values.
The bond lengths around the metal centre observed in the X-ray crystal structures were accurately reproduced, as well as the ligand bite angle. The estimation of trigonal twist in X-ray crystallographic structures was less successful, with the
molecular model overestimating this by on average 5.4°. The model was more successful in predicting the quantum mechanical energies required to deform simple ferric-tris-hydroxamates by stretching and rotation of the ligands – the latter to imitate trigonal distortion of the complexes.