A structural and functional analysis of type III periplasmic and substrate binding proteins: their role in bacterial siderophore and heme transport.
ABSTRACT In Escherichia coli the Fhu, Fep and Fec transport systems are involved in the uptake of chelated ferric iron-siderophore complexes, whereas in pathogenic strains heme can also be used as an iron source. An essential step in these pathways is the movement of the ferric-siderophore complex or heme from the outer membrane transporter across the periplasm to the cognate cytoplasmic membrane ATP-dependent transporter. This is accomplished in each case by a dedicated periplasmic binding protein (PBP). Ferric-siderophore binding PBPs belong to the PBP protein superfamily and adopt a bilobal type III structural fold in which the two independently folded amino and carboxy terminal domains are linked together by a single long α-helix of approximately 20 amino acids. Recent structural studies reveal how the PBPs of the Fhu, Fep, Fec and Chu systems are able to bind their corresponding ligands. These complex structures will be discussed and placed in the context of our current understanding of the entire type III family of Gram-negative periplasmic binding proteins and related Gram-positive substrate binding proteins.
- SourceAvailable from: Hans Jörg Kunte
Dataset: PNAS-2011-Marinelli-E1285-92 copy
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ABSTRACT: Escherichia coli HisJ is a type II periplasmic binding protein (PBP) that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein of which the domain 1 (D1) is made up of two non-contiguous subdomains and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2 we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, while D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, while isolated D2 experiences a significant reduction in slower (ms-μs) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact in the presence or absence of histidine, which indicates the importance of intact interdomain connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II PBPs where ligand is initially bound by D1 and D2 plays a supporting role in this dynamic process.Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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ABSTRACT: Lipoprotein FtsB is a component of the FtsABCD transporter that is responsible for ferrichrome binding and uptake in the Gram-positive pathogen Streptococcus pyogenes. In the present study, FtsB was cloned and purified from the bacteria and its Fch binding characteristics were investigated in detail by using various biophysical and biochemical methods. Based on the crystal structures of homogeneous proteins, FtsB was simulated to have bi-lobal structure forming a deep cleft with four residues in the cleft as potential ligands for Fch binding. With the assistance of site-directed mutagenesis, residue Trp204 was confirmed as a key ligand and Tyr137 was identified to be another essential residue for Fch binding. Kinetics experiments demonstrated that Fch binding in FtsB occurred in two steps, corresponding to the bindings to Tyr137 at N-lobe and Trp204 from C-lobe, respectively, and so that closing the protein conformation. Without either residue Tyr137 or Trp204, Fch binding in the protein as mutants Fch-Y137A and Fch-W204A may have a loose conformation, resembling the apo-proteins in proteolysis resistance and migration behaviors in native gel. This study revealed the inconsistence in the key amino acids among Fch-binding proteins from Gram-positive and -negative bacteria, providing interesting findings for understanding the differences between Gram-positive and -negative bacteria in the mechanism of iron uptake via siderophore (Fch) binding and transport.PLoS ONE 01/2013; 8(6):e65682. · 3.73 Impact Factor