Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

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. Bacteria are dependent upon effective and efficient iron acquisition mechanisms to survive and proliferate in the ironlimited environment of the host (1-3). Pathogenic Gram-negative bacteria within the Neisseriaceae and Pasteurellaceae families rely on specialized uptake systems to acquire iron directly from host iron-binding proteins (2-5). These bacteria can acquire iron from host transferrin (Tf) 3 in particular (6), but in some cases also lactoferrin (7, 8) and hemogobulin/haptoglobulin (9). To understand such uptake mechanisms, a detailed characterization of relevant receptor-host protein interactions is required.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). They have been found in clinical isolates of the Neisseriaceae, Pasteurellaceae, as well as the Moraxellaceae families (12) and constitute the outer membrane receptor complex responsible for binding Tf and transporting iron across the outer membrane into the periplasmic space (4, 13). These receptors exhibit a strict host specificity. For example, receptors of porcine pathogens will specifically bind porcine transferrin (pTf) but not human, avian, bovine, or ovine Tf (14).Tf is a glycoprotein of ϳ80 kDa, composed of two highly homologous N and C lobes (15). Each lobe contains two domains, connected by two antiparallel ␤-strands in a clamshell-like fold, producing a cleft that coordinates Fe 3ϩ in the binding pocket, along with a synergistic anion (CO 3 2Ϫ or C 2 O 4 ...

High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio

“…With the incorporation of restraints from two data sources, the Haddock-generated model shows an interaction between TbpB and the two C-lobes of pTf, as expected (41). These lobes close around Fe 3ϩ in the iron-loaded holo form, bringing them into registry with the TbpB binding surface.…”

“…Structures for the individual elements of the complex have been determined (pTf: 1H76 and H49 TbpB: 3HOL), and a RosettaDock model of the binary complex has been validated by extensive mutational analysis and functional studies (Fig. 5A) (41,50). A crystallographic structure of human transferrin bound to TbpB from Neisseria meningitidis provides strong supporting evidence for the localization of the binding site (51).…”

“…Models of the pTf:TbpB interaction. A, Docked model of 1H76 (pTf) and 3HOL (TbpB) using modified routines in RosettaDock, as published and validated through mutational analysis of key residues in the binding interface (41,50). Crosslinks detected by the Studio plug-in are highlighted in red bars, with a linking scheme (and distances) shown on the right.…”

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

“…The present study focused on receptor proteins from A. pleuropneumoniae due to the availability of information on the TbpB-Tf interaction [16,17] and success with expressing and isolating TbpA from this species. Using H/DX-MS to map the interaction with TbpA required a revised protocol, to remove the detergent prior to mass analysis.…”

“…Thus a key question regarding TbpB function is whether it simply delivers holo-Tf to TbpA or whether it assists in the iron acquisition process by jointly acting on Tf with TbpA. In order to understand if and how TbpB and TbpA function synergistically, this was compared with the TbpB footprint [17,18], and affinitycapture approaches were used to determine whether TbpB and TbpA can simultaneously bind to Tf. In the present study we have adapted new methodologies for rapidly removing detergents under the stringent requirements of H/DX-MS (H/DX coupled to MS) [29], and subsequently mapped the effect of TbpA on Tf.…”