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

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.


(a) Crystal structure of H227L-Fe-5-Lic with the electron density for the maximum likelihood weighted map contoured at the 2σ level. The Y288 and L227 sidechains are shown in cylinders with carbon atoms in gold. Fe(III) atom is shown as a gray sphere, and the 5-Lic ligand in ball and stick colored by atom type. L227 is located away from the binding pocket, with a water molecule (red sphere) completing the octahedral coordination sphere. (b) Alternate view of crystal structure of H227L-Fe-5-Lic showing hexacoordinate Fe(III) with a water ligand.
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f5: (a) Crystal structure of H227L-Fe-5-Lic with the electron density for the maximum likelihood weighted map contoured at the 2σ level. The Y288 and L227 sidechains are shown in cylinders with carbon atoms in gold. Fe(III) atom is shown as a gray sphere, and the 5-Lic ligand in ball and stick colored by atom type. L227 is located away from the binding pocket, with a water molecule (red sphere) completing the octahedral coordination sphere. (b) Alternate view of crystal structure of H227L-Fe-5-Lic showing hexacoordinate Fe(III) with a water ligand.

Mentions: All five mutants (Y288F, H227L, H227A, H227L/Y288F, H227A/Y288F) were crystallized in their apo form, and all structures are closely similar to that of wild type CeuE. All mutations were confirmed by sequencing of the DNA. However while there is clear electron density for the Y288F mutation in its three structures, H227L and H227A are located in a flexible loop region and there is generally no electron density to confirm their presence. Co-crystallization of mutant H227L with Fe-5-Lic yielded a structure containing three protein ligand complexes in the asymmetric unit (PDB ID: 5TCY). The loop region upon which the H227L mutation is ordered, but points away from the Fe(III) coordination sphere. A water molecule completes the octahedral Fe(III) coordination (with good density in only one of the three independent molecules in the asymmetric unit, Fig. 5).


Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM 4 − siderophore analogues of varied linker length
(a) Crystal structure of H227L-Fe-5-Lic with the electron density for the maximum likelihood weighted map contoured at the 2σ level. The Y288 and L227 sidechains are shown in cylinders with carbon atoms in gold. Fe(III) atom is shown as a gray sphere, and the 5-Lic ligand in ball and stick colored by atom type. L227 is located away from the binding pocket, with a water molecule (red sphere) completing the octahedral coordination sphere. (b) Alternate view of crystal structure of H227L-Fe-5-Lic showing hexacoordinate Fe(III) with a water ligand.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: (a) Crystal structure of H227L-Fe-5-Lic with the electron density for the maximum likelihood weighted map contoured at the 2σ level. The Y288 and L227 sidechains are shown in cylinders with carbon atoms in gold. Fe(III) atom is shown as a gray sphere, and the 5-Lic ligand in ball and stick colored by atom type. L227 is located away from the binding pocket, with a water molecule (red sphere) completing the octahedral coordination sphere. (b) Alternate view of crystal structure of H227L-Fe-5-Lic showing hexacoordinate Fe(III) with a water ligand.
Mentions: All five mutants (Y288F, H227L, H227A, H227L/Y288F, H227A/Y288F) were crystallized in their apo form, and all structures are closely similar to that of wild type CeuE. All mutations were confirmed by sequencing of the DNA. However while there is clear electron density for the Y288F mutation in its three structures, H227L and H227A are located in a flexible loop region and there is generally no electron density to confirm their presence. Co-crystallization of mutant H227L with Fe-5-Lic yielded a structure containing three protein ligand complexes in the asymmetric unit (PDB ID: 5TCY). The loop region upon which the H227L mutation is ordered, but points away from the Fe(III) coordination sphere. A water molecule completes the octahedral Fe(III) coordination (with good density in only one of the three independent molecules in the asymmetric unit, Fig. 5).

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.