Kinetic analysis of ligand interaction with the gonococcal transferrin-iron acquisition system

Abstract

The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the Cterminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.

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

“…Even with the C-terminal portion of the anchor peptide wrapped around the C-terminal lobe, the Tf-binding cap region of the N-terminal lobe could be positioned Ͼ60 Å from the outer membrane surface. The observation that maximal binding of labeled Tf by intact cells was attained at the earliest measured time point in a TbpBϩve, TbpAϪve strain, but not in a TbpBϪve, TbpAϩve strain (16), is consistent with a model in which TbpB is fully accessible at the cell surface. Because the intact anchor peptide does not mediate binding of TbpB to TbpA in the absence of Tf (Fig.…”

“…A conformational change related to Tf binding might account for the nearly irreversible binding to TbpAs (Fig. 2 and Table 1) and may relate to the observation that the efficient release of bound Tf from TbpA at the gonococcal cell surface required the presence of TonB (16). In the TbpBϪve, TonBϪve strains, there was no detectable displacement of the bound, labeled Tf by unlabeled Tf after a 40-min incubation when compared with Ͼ60% released in the TonB-proficient strain.…”

“…The protection from proteolysis of TbpB by TbpA is dependent upon interaction with TonB (15), suggesting that the TbpB-TbpA interaction is linked to the iron acquisition process. Similarly, the conclusion that TbpA and TbpB "work together" during the association phase of binding radiolabeled Tf (16) implies that there is an intimate association between the receptors during the Tf binding step. However, experiments with gold-labeled antibodies in electron micrograph experiments demonstrated that TbpA and TbpB are not co-localized in the absence of added Tf (17), suggesting that they do not normally constitute a preformed receptor complex in the membrane.…”

Structural insight into the lactoferrin receptors from pathogenic Neisseria

“…It was further proposed that in addition to TbpB's role in specifically binding and concentrating iron-loaded transferrin at the neisserial surface, it may also serve to lock transferrin in the closed iron-bound state until delivery to TbpA for iron extraction and import, thereby increasing the efficiency of the transferrin-iron import mechanism. TbpB has been reported to also participate in both iron extraction from transferrin and the release of apo-transferrin from TbpA following iron transport; however, the exact mechanisms for these roles remain elusive (DeRocco et al, 2009; Siburt et al, 2009). …”

Transition metals at the host–pathogen interface: howNeisseriaexploit human metalloproteins for acquiring iron and zinc

“…TpbA binds apo- and holo-hTF with similar affinities, and extracts Fe from hTF in the absence of TpbB. Nevertheless, iron removal from holo-hTF occurs more rapidly when the co-receptor is present [51,52]. TbpB preferentially binds holo-hTF and thereby facilitates capture of the holo-form and, following Fe(III) extraction, release of apo-hTF [53,54].…”