Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4\u2212 siderophore analogues of varied linker length

Daniel J Raines,Johan P Turkenburg,Olga V Moroz,Ross P Thomas,Ellis J Wilde,Adam Hughes,Anne-Kathrin Duhme-Klair,Elena V Blagova,Keith S Wilson

doi:10.1038/srep45941

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.

Highlights

The optimum linker length of five corresponds to that found in natural siderophores that contain lysine or di-serine-based linkers, such as enterobactin and azotochelin
Circular dichroism indicated that the Λ-configured ligand arrangement observed in the crystal structure is maintained in solution phase, and that the Λ-configuration is selected by H227L for all Fe-n-Lic species, but with a weaker signal than for wild type CeuE, due to weaker binding affinities across the board
The numbering used for C. jejuni CeuE starts at the first residue of the mature protein after cleavage of the 20 amino acid signal peptide[20]

Summary

Introduction

Circular dichroism indicated that the Λ-configured ligand arrangement observed in the crystal structure is maintained in solution phase, and that the Λ-configuration is selected by H227L for all Fe-n-Lic species, but with a weaker signal than for wild type CeuE, due to weaker binding affinities across the board. The mutant proteins were expressed and purified according to the standard procedure detailed for wild type CeuE. For co-crystallization, a solution of CeuE was diluted to 20 mg mL−1 in a buffer of 20 mM Tris, 150 mM NaCl at pH 7.5.

Results

Conclusion