Abstract
Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.
Highlights
The bacterial Tf receptors are composed of two iron-repressible surface components, transferrin binding protein A (TbpA), a TonB-dependent integral outer membrane protein of ϳ100 kDa, and transferrin-binding protein B (TbpB), a lipoprotein varying in size from 60 to 100 kDa [2, 3, 5, 10, 11]
Recent work by Ling et al has described two TbpB variants from Neisseria meningitidis that interact with the C lobe of human transferrin [11], it has been previously reported that both Tf lobes are involved in binding, JUNE 17, 2011
Together with a companion study by Calmettes et al [31], we provide a structural analysis of the receptor-Tf interaction representing several divergent porcine pathogenic strains and evaluate the impact of iron loading on the molecular recognition mechanism in greater detail
Summary
Production of Recombinant TbpB—The mature coding region of TbpB from Actinobacillus pleuropneumoniae strain ApH49 (serotype7), ApH87 (serotype 5), ApH89 (serotype 1) and Actinobacillus suis AsH57 were PCR-amplified and cloned in to the in-frame BamHI restriction site of a customized expression vector preceded by a polyhistidine region, a maltose-binding protein (Mbp) and a tobacco etch virus protease cleavage site. Mass Shift Analysis by H/DX—Each H/DX experiment involved a measurement of deuteration levels for pTf in the presence and absence of each of the four TbpB receptors. In this fashion, changes in deuteration arising from complexation could be used to identify the binding interface. Changes in deuteration arising from complexation could be used to identify the binding interface These measurements were made using mass spectrometry of pTf peptides, generated by digesting deuterated protein under conditions preventing back-exchange of deuterium. Absorption Studies of FbpA—Apo-FbpA was incubated with pTf-TbpB complex as described in the SUPREX experiment. The remaining free iron was removed via gel filtration chromatography, and the iron-loaded FbpA (apo-FbpA ϩ FeCl3) was concentrated using an Amicon concentrator as described
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