Interdomain interactions between the hydrophilic domains of the mannitol transporter of Escherichia coli in the unphosphorylated and phosphorylated states

Interdomain interactions in the mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli play a key role in the mechanism of mannitol transport across the membrane [Beer et al. (1995) Biochemistry 34, 3239-3247; Lolkema et al, (1991) Biochemistry 30, 6716-6721]. In this study, we focus on the interactions between the hydrophilic A and B domains and try to determine those as a function of the phosphorylation state of the enzyme. To this end, unfolding studies on the subcloned domains IIA(mtl) and IIBmtl, as well as on the binary combination IIBA(mtl), were performed, both in the unphosphorylated and in the phosphorylated states, using GuHCl and heat as the denaturant, It is shown that IIA(mtl) and IIBmtl, as well as P-IIA(mtl) and P-IIBmtl, unfold according to a two-state mechanism but that IIBA(mtl) and P2-IIBA(mtl) do not exhibit such behavior, Two transitions are observed instead, indicating a lack of strong positive cooperative interactions. DSC studies of the unphosphorylated proteins showed a destabilization of the B domain in IIBA(mtl) with respect to the free IIBmtl as indicated by a lowering of the melting temperature and a lower enthalpy of unfolding, Furthermore, it is shown that phosphorylation has a destabilizing effect on both IIA(mtl) and IIBA(mtl) but not on IIBmtl. Possible explanations for this behavior and the biological relevance of the destabilizing forces in IIBA(mtl) are discussed.