Insight into the Structure and Function of the Transferrin Receptor from Neisseria meningitidis Using Microcalorimetric Techniques*

The transferrin receptor of Neisseria meningitidis is composed of the transmembrane protein TbpA and the outer membrane protein TbpB. Both receptor proteins have the capacity to independently bind their ligand human transferrin (htf). To elucidate the specific role of these proteins in receptor function, isothermal titration calorimetry was used to study the interaction between purified TbpA, TbpB or the entire receptor (TbpA + TbpB) with holo- and apo-htf. The entire receptor was shown to contain a single high affinity htf-binding site on TbpA and approximately two lower affinity binding sites on TbpB. The binding sites appear to be independent. Purified TbpA was shown to have strong ligand preference for apo-htf, whereas TbpA in the receptor complex with TbpB preferentially binds the holo form of htf. The orientation of the ligand specificity of TbpA toward holo-htf is proposed to be the physiological function of TbpB. Furthermore, the thermodynamic mode of htf binding by TbpB of isotypes I and II was shown to be different. A protocol for the generation of active, histidine-tagged TbpB as well as its individual N- and C-terminal domains is presented. Both domains are shown to strongly interact with each other, and isothermal titration calorimetry and circular dichroism experiments provide clear evidence for this interaction causing conformational changes. The N-terminal domain of TbpB was shown to be the site of htf binding, whereas the C-terminal domain is not involved in binding. Furthermore, the interactions between TbpA and the different domains of TbpB have been demonstrated.

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