Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force

Abstract

SummaryWe have characterized the interaction of the Neisseria meningitidis TonB-dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [

Analysis of Haptoglobin and Hemoglobin-Haptoglobin Interactions with theNeisseria meningitidisTonB-Dependent Receptor HpuAB by Flow Cytometry

“…In those experiments, wildtype HpuAB saturably bound 125 I-Hb with a moderate affinity approximately 50-fold lower than that of the interaction of Tf with the TbpAB receptor (15,69). We did not detect significant Hb binding to mutant strains lacking either HpuA, HpuB, or both receptor components by using that assay or a solidphase dot blot analysis (69). Based on those observations, we concluded that both HpuA and HpuB were required to bind and to utilize Hb.…”

“…No known homologues of HpuA exist in public databases, and its role in HpuAB function remains to be determined. We have reported data suggesting that HpuA and HpuB physically interact to form a functional receptor complex (51,69). Characterization of isogenic single mutants of HpuA and HpuB suggested that both receptor components are absolutely required for the utilization of Hb or HbHp (48,69).…”

“…This model was based on the highly conserved three-dimensional X-ray crystallographic structures of three E. coli TonB-dependent siderophore receptors: FepA, FhuA, and FecA (6,(23)(24)(25)52). The 35-kDa HpuA accessory lipoprotein is peripherally associated with the OM via an N-terminally linked fatty acid tether (49,69). No known homologues of HpuA exist in public databases, and its role in HpuAB function remains to be determined.…”

“…However, Cornelissen and Sparling reported that solid-phase assays did not accurately represent the binding of ligand to live cells, perhaps because receptor conformations important for ligand binding were disrupted by the drying process (15). Therefore, we had previously developed a liquid-phase 125 I-Hb binding assay to measure the equilibrium binding kinetics of Hb to HpuAB on live meningococci (69). In those experiments, wildtype HpuAB saturably bound 125 I-Hb with a moderate affinity approximately 50-fold lower than that of the interaction of Tf with the TbpAB receptor (15,69).…”

“…Therefore, we had previously developed a liquid-phase 125 I-Hb binding assay to measure the equilibrium binding kinetics of Hb to HpuAB on live meningococci (69). In those experiments, wildtype HpuAB saturably bound 125 I-Hb with a moderate affinity approximately 50-fold lower than that of the interaction of Tf with the TbpAB receptor (15,69). We did not detect significant Hb binding to mutant strains lacking either HpuA, HpuB, or both receptor components by using that assay or a solidphase dot blot analysis (69).…”