Transferrin binding and iron utilization by N. gonorrhoeae is a complex process involving at least three proteins, TbpA, TbpB and TonB. This process incorporates four steps: Tf binding, removal of iron from Tf, internalization of iron, and release of Tf from the cell surface. TbpA has the ability to complete all four of these steps; however, it requires TonB to accomplish iron internalization and subsequent Tf release. The methodologies employed in the current study do not allow us to determine whether TonB-derived energy is necessary for removal of iron from Tf.
Examination of Tf association with the wild-type cell surface indicated that both TbpA and TbpB work together to complete this step, as only one phase of Tf association was detected. Cornelissen et al. suggested that TbpB undergoes a TbpA-dependent conformational change (Cornelissen, Anderson et al. 1997
). Therefore, TbpB may behave differently in the presence of the outer membrane transporter, an hypothesis supported by our association experiments. Tf binding to TbpA was enhanced by the presence of TonB. In contrast, ligand association with the wild-type system, comprised of both proteins, was apparently TonB independent, further supporting the conclusion that both TbpA and TbpB function synergistically during the initial step of Tf-iron acquisition.
The crystal structure of FepA, a TonB-dependent transporter, indicates that the surface exposed loops are disordered, implying flexibility of these domains (Buchanan, Smith et al. 1999
). Additionally, it was demonstrated that the loops of FepA move upon binding and transport of the ligand (Klug, Eaton et al. 1998
; Scott, Newton et al. 2002
). Presumably, TbpA undergoes a similar conformational change to accomplish iron stripping and transport. Insertion into the surface exposed loop 9 of TbpA rendered a utilization-deficient mutant (Yost-Daljev and Cornelissen 2004
). Transferrin rapidly and completely dissociated from this strain, in contrast to the parental strain expressing wild-type TbpA. This result implies that the slower off-rates demonstrated by wild-type TbpA may result from internalization of Tf-bound iron.
Tf dissociation from TbpB occurred in a biphasic manner, consistent with the presence of two binding sites. The majority of Tf was released from TbpB rapidly, as approximately 60% dissociated within 1 minute. The results from the current study suggested that the C-lobe of TbpB was required for the rapid release of Tf. It has been demonstrated that the C-lobe of Tf specifically associates with the N-lobe of TbpB while the N-lobe of the ligand binds to the C-lobe of TbpB (Retzer, Yu et al. 1999
; Sims and Schryvers 2003
). This binding pattern suggests that each domain of TbpB interacts with a single lobe of Tf (Alcantara and Schryvers 1996
), which results in a strengthened interaction between receptor and ligand. Previous studies have suggested that each half of TbpB wraps around an individual lobe of Tf. Therefore, TbpB undergoes extensive conformational changes upon interaction with the ligand (Retzer, Yu et al. 1999
). Our results from ligand binding and kinetic studies support this contention, indicating that the two Tf binding sites of TbpB create one binding pocket for the ligand.
The TbpB-HA fusion proteins demonstrated a single binding site with an affinity for Tf of approximately 10nM, in agreement with previous studies (Cornelissen and Sparling 1996
). Three TbpB-HA mutants contain insertions in a Tf binding domain of TbpB: MCV815 (HA4), MCV817 (HA5) and MCV823 (HA8) (DeRocco and Cornelissen 2007
). The insertions into MCV815 and MCV816 interrupted the N-terminal Tf binding domain of TbpB, while the insertion in MCV823 has impacted the C-terminal ligand-binding domain (DeRocco and Cornelissen 2007
). Under the conditions tested here, 125
I-Tf binding could not be detected in the Tf binding domain mutants (data not shown). Our previous studies indicated that these strains demonstrate reduced but specific binding of Tf in solid phase binding assays (DeRocco and Cornelissen 2007
). It has been suggested that the individual domains of TbpB do not bind Tf to the same degree as the wild-type protein (Renauld-Mongenie, Poncet et al. 1997
; Retzer, Yu et al. 1999
; Krell, Renauld-Mongenie et al. 2003
), implying that each half of TbpB has a much lower affinity for the ligand as compared to the full-length protein. We hypothesize that by interrupting either binding domain in TbpB, the remaining bound Tf could not be detected in the assays used in the current study. In an attempt to address this possibility we increased the ligand incubation time as well the concentration of 125
I-Tf. Despite these alterations, binding was not detected for any of the strains. Lower affinity binding can be difficult to detect using rapid filtration assays as the ligand quickly dissociates during the washing and filtration steps (Jones 1982
; Bylund and Yamamura 1990
; Jian-Xin Wang 1992
). Collectively, these results support the idea that the individual domains of TbpB have a lower affinity for Tf as compared to the full-length protein and therefore both domains are necessary to achieve wild-type levels of binding.
TonB plays a role in the kinetics of Tf binding to TbpA, but does not directly impact TbpB. We hypothesize that in energizing TbpA, TonB causes a conformational change in the transporter, and by elimination of this interaction TbpA remains static and unable to accomplish iron transport. However, once TbpA interacts with TonB, this receptor can carry out the remaining steps in Tf-iron acquisition. In this model, once TbpA has removed iron from the ligand, TbpA cannot release and bind to another molecule of Tf until iron transport into the periplasm has been accomplished with the participation of TonB.
TbpB is the only non-essential component of the Tf-iron acquisition system. The role of this lipoprotein in Tf utilization has not been completely defined; however TbpB makes iron uptake from Tf more efficient (Anderson, Sparling et al. 1994
). Our analyses demonstrate that this increase in efficiency is in part due to the ability of TbpB to affect rapid association and dissociation of Tf with the cell surface. Since TbpB exhibits holo-Tf binding specificity, it is possible that this lipoprotein serves to deliver the optimum ligand to TbpA and once the iron has been removed from the ligand, to aid in the release of apo-Tf. This model is supported by the observation that TbpA did not efficiently release Tf in the absence of TbpB. Once iron has been removed from Tf it is necessary to quickly release the apo, iron-depleted ligand, and the C-lobe of TbpB potentially contributes to this task. Since TbpA, unlike TbpB, shows no specificity for holo-Tf (Cornelissen and Sparling 1996
), the C-terminus of TbpB may also function to rapidly remove the apo-ligand.
This study highlights the individual roles of the proteins involved in the Tf-iron acquisition system. In our current model, TbpA, although capable of releasing Tf in the absence of TbpB, accomplishes this at a dramatically slower rate. Previous data demonstrated that a TbpA-only strain internalized 50% less iron from Tf as compared to the wild-type strain (Anderson, Sparling et al. 1994
). This result, along with ligand dissociation data from the current study, suggests that the transporter removes iron from Tf but cannot efficiently release the apo-ligand from the cell surface. Therefore, overall iron internalization is reduced in a TbpA-only strain. We conclude that TbpB contributes to increased Tf-iron acquisition efficiency by affecting rapid association and dissociation of the optimum ligand so that multiple rounds of iron internalization can occur.