Anchor Peptide of Transferrin-binding Protein B Is Required for Interaction with Transferrin-binding Protein A

Xue Yang,Rong-Hua Yu,Charles Calmettes,Trevor F Moraes,Anthony B Schryvers

doi:10.1074/jbc.m110.214171

Abstract

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.

Highlights

Transferrin receptors are critical for survival of important Gram-negative bacterial pathogens
Preliminary analysis of expression indicated that the highest levels were achieved with their respective mobile phase buffer (TbpA) from A. pleuropneumoniae, and considering the prior success with producing and crystallizing Tf-binding protein B (TbpB) from this species [18, 19], we decided to focus our studies on receptor proteins from this species
The original isolation of two Tf receptor proteins by affinity capture experiments [28] and the early experiments demonstrating that both proteins were required for growth on Tf as an iron source [5] led to an initial view that they formed a functional receptor complex responsible for binding Tf at the cell surface and removing iron that was transported into the cell

Summary

Background

Transferrin receptors are critical for survival of important Gram-negative bacterial pathogens. Gram-negative pathogenic bacteria in the Neisseriaceae, Moraxellaceae, and Pasteurellaceae families are major causes of important infections of humans (meningitis, pneumonia, otitis media, gonorrhea) and food production animals (shipping fever in cattle, pneumonia in pigs) They primarily reside in the respiratory and genitourinary tract of their hosts and rely on a specialized iron uptake system that acquires iron from serum transferrin (Tf).. The interaction of TbpB with holoTf involves regions on both the C1 and the C2 domains of the C-lobe [20, 21] maintaining Tf in the closed conformation, limiting the release of iron [20, 23] It is unclear how TbpA is capable of binding both the holo and the apo forms of Tf [12], but it might suggest that TbpA binds both forms of Tf in an intermediate conformation. This study was initiated to investigate the TbpB-TbpA interaction and gain further insights into the role of TbpB in the iron acquisition process

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION