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 [Boer et al. (1995) Biochemistry 34, 3239-3247; Loikema et al. (1991) Biochemistry 30, 6716-6721]. In this study, we focus on the interaction 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 IIAmtl and IIBmtl, as well as on the binary combination IIBAmtl, were performed, both in the unphosphorylated and in the phosphorylated states, using GuHCl and heat as the denaturant. It is shown that IIAmtl and IIBmtl, as well as P-IIAmtl and P-IIBmtl, unfold according to a two-state mechanism but that IIBAmtl and P2-IIBAmtl 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 IIBAmtl with respect to the free IIBmtl as indicated by a lowereing of the melting temperature and a lower enthalpy of unfolding. Furthermore, it is shown that phosphorylation has a destablilizing effect on both IIAmtl and IIBAmtl but not on IIBmtl. Possible explanations for this behavior and the biological relevance of the destabilizing forces in IIBAmtl are discussed.
Meijberg
W
W
Department of Biochemistry, University of Groningen, Groningen, The Netherlands.
Schuurman-Wolters
G K
GK
Robillard
G T
GT
D001665:Binding Sites; D001693:Biological Transport, Active; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000378:metabolism*; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli