Abstract
The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-angstroms structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure.
Highlights
Metal ions are indispensable components of biological systems
Spectral analysis of the protein preparations demonstrated that visible absorption maximum at 513 and 510 nm wavelengths for YfuA and SfuA, respectively, contrasting the maximum at 480 nm for the H. influenzae FbpA (HiFbpA)
The C␣ backbones of these proteins are virtually superimposable, our findings suggest that YfuA and SfuA represent a new class in this iron-binding family of proteins based on metal coordination (Fig. 4)
Summary
Cloning and Purification—The intact sfuA gene was amplified from a clinical isolate of S. marcescens obtained from the Foothills Hospital (Calgary, Alberta) with a forward primer introducing an NdeI site at the start codon preceded immediately by a BclI site and with a reverse. Crystals used for diffraction analysis were grown at 4 °C for SfuA and at 20 °C for YfuA by the sitting drop vapor-diffusion technique, with 2-l drops (1 l of protein sample and 1 l of reservoir solution) and an 85-l reservoir. The YfuA crystal was dipped into crystallization mother liquor supplemented with 25% (v/v) ethylene glycol The crystals of both proteins were maintained at 100 K during data collection. To solve the YfuA structure, single wavelength anomalous diffraction x-ray data were analyzed using the program SHELXD [29] to find the heavy atom substructure, locating the six sulfur atoms from methionine residues, the iron, and five bound zinc atoms. The resulting experimental phases were of sufficient quality to allow automated building of almost the entire protein structure in electron density with the program ARP/wARP [32]. During the later stages of refinement, difference maps (Fo Ϫ Fc maps) were used to place the bound iron, zinc, carbonate, ethylene glycol, and water molecules
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