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Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM 4 − siderophore analogues of varied linker length

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ABSTRACT

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4−) to five, six and eight (5-, 6-, 8-LICAM4−, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4− structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4− is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4− but decreases for 6- and 8-LICAM4−. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

No MeSH data available.


Selected fluorescence quenching data for binding to (a) CeuE (b) mutant H227L and (c) mutant H227A. Fe-4-Lic (red circles), Fe-5-Lic (gold squares), Fe-6-Lic (blue inverted triangles) and Fe-4-Lic (green triangles) (240 nM protein in 40 mM TrisHCl pH 7.5, NaCl 150 mM).
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f6: Selected fluorescence quenching data for binding to (a) CeuE (b) mutant H227L and (c) mutant H227A. Fe-4-Lic (red circles), Fe-5-Lic (gold squares), Fe-6-Lic (blue inverted triangles) and Fe-4-Lic (green triangles) (240 nM protein in 40 mM TrisHCl pH 7.5, NaCl 150 mM).

Mentions: The affinities of CeuE for the four Fe-n-Lic complexes were quantified using intrinsic fluorescence quenching, Table 1, with measurements carried out in triplicate. The data were fitted to a 1:1 binding model and analyzed by non-linear regression26. Whilst the binding of Fe-5-Lic was too tight to allow the calculation of an accurate dissociation constant, a comparison of the shape of the individual titration curves suggest that Fe-5-Lic binds slightly more tightly than either Fe-4-Lic or Fe-6-Lic (Fig. 6(a)). An attempt to determine the Kd value of Fe-5-Lic by using a more dilute protein solution was unsuccessful because of the inherent decrease in emission intensity reaching the detection limit of the fluorimeter.


Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM 4 − siderophore analogues of varied linker length
Selected fluorescence quenching data for binding to (a) CeuE (b) mutant H227L and (c) mutant H227A. Fe-4-Lic (red circles), Fe-5-Lic (gold squares), Fe-6-Lic (blue inverted triangles) and Fe-4-Lic (green triangles) (240 nM protein in 40 mM TrisHCl pH 7.5, NaCl 150 mM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382913&req=5

f6: Selected fluorescence quenching data for binding to (a) CeuE (b) mutant H227L and (c) mutant H227A. Fe-4-Lic (red circles), Fe-5-Lic (gold squares), Fe-6-Lic (blue inverted triangles) and Fe-4-Lic (green triangles) (240 nM protein in 40 mM TrisHCl pH 7.5, NaCl 150 mM).
Mentions: The affinities of CeuE for the four Fe-n-Lic complexes were quantified using intrinsic fluorescence quenching, Table 1, with measurements carried out in triplicate. The data were fitted to a 1:1 binding model and analyzed by non-linear regression26. Whilst the binding of Fe-5-Lic was too tight to allow the calculation of an accurate dissociation constant, a comparison of the shape of the individual titration curves suggest that Fe-5-Lic binds slightly more tightly than either Fe-4-Lic or Fe-6-Lic (Fig. 6(a)). An attempt to determine the Kd value of Fe-5-Lic by using a more dilute protein solution was unsuccessful because of the inherent decrease in emission intensity reaching the detection limit of the fluorimeter.

View Article: PubMed Central - PubMed

ABSTRACT

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4−) to five, six and eight (5-, 6-, 8-LICAM4−, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4− structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4− is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4− but decreases for 6- and 8-LICAM4−. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

No MeSH data available.