The Thermal Stability and Domain Interactions of the Mannitol Permease of Escherichia coli

Abstract

The transport of some carbohydrates from the environment into bacterial cells is mediated by the proteins from the phosphoenolpyruvate-dependent phosphotransferase system (PTS). 1This system couples the translocation of substrates across the cytoplasmic membrane to concomitant phosphorylation, using phosphoenolpyruvate (PEP) as the energy source. Thus, the overall reaction catalyzed by the PTS system is best described as follows.PEP ϩ carbohydrate out 7 pyruvate ϩ P-carbohydrate in REACTION 1The equilibrium of this reaction is shifted far to the right.The transport proteins of the PTS, termed enzyme IIs, 2 are specific for the carbohydrate they transport but nonetheless show a remarkably similar architecture throughout. In almost all systems, a membrane-spanning domain and two cytoplasmic phosphotransfer domains are found, in some cases covalently bound and in other cases as separate proteins or as combinations of these two possibilities. The phosphoryl group is transferred to the first of the phosphotransfer domains or proteins, the A domain, via two general PTS proteins, enzyme I and HPr, and from there to the second phosphotransfer domain, the B domain. The incoming substrate is bound by the C domain, translocated, and phosphorylated directly by the B domain. Reviews on the PTS systems can be found in Refs. 1-5.The subject of this study is the mannitol-specific PTS protein from E. coli, enzyme II mtl . In this protein, the three domains are all part of the same polypeptide chain, with the C domain at the N terminus and the A domain at the C terminus. The phosphorylation sites are His 554 on the A domain and Cys 384 on the B domain. All three domains as well as the binary combinations, IICB mtl and IIBA mtl , have been subcloned and overexpressed separately and were shown to be enzymatically active in the presence of the other constituents of the PTS (6 -10).In contrast to the large number of studies on the stability of water-soluble proteins, comparatively little is known about the factors determining the stability of membrane proteins in their natural environment. This is mostly caused by the fact that membrane proteins are more difficult to handle than their water-soluble counterparts due to their inherent hydrophobicity, often resulting in aggregation and/or precipitation. The effect is even stronger in the unfolded state, since unfolding results in exposure to the solvent of hydrophobic parts that are normally buried in the interior of the protein. In this study, we circumvented this problem by reconstituting EII mtl , as well as IICB mtl and IIC mtl , in a lipid bilayer, enabling us to study the thermal stability of the enzyme by differential scanning calorimetry and CD spectroscopy.Data from unfolding studies can also be used to obtain information on the extent of the interactions between the domains in a protein. The energy required to take the enzyme from its

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

“…4 and Table V). DSC profiles showing two separate unfolding transitions have been observed in the past for proteins containing two separate domains (40,41). The C-terminal domain was found to be fairly thermostable with a T m beyond 80°C, which is astonishing considering the relatively low amount of secondary structure determined by cd spectroscopy (Fig.…”

Analysis of the Interaction between the Global Regulator Mlc and EIIBGlc of the Glucose-specific Phosphotransferase System in Escherichia coli

“…The existence of such mutations implies a tight coupling between sugar transport through EIIC Glc , the phosphorylation by EIIB Glc , and the conformation around Arg-424 and Cys-421. A conformational coupling between the IIC and IIB domains has been detected previously in the mannitol enzyme IICBA transporter (34).…”

Cysteine Cross-linking Defines Part of the Dimer and B/C Domain Interface of the Escherichia coli Mannitol Permease

“…SSCS and SSCS-S124C were purified by Ni-NTA-agarose affinity chromatography as described for 6HEII mtl (14), except that dPEG was used as the detergent. IIChis-CL and IIChis-S124C were purified as described (16).…”

“…5) Isothermal titration calorimetry experiments indicated that a significant part of the structural changes upon the binding of mannitol to the C domain reside in the B domain. Approximately 50 -60 residues are removed from the bulk water upon binding of mannitol, which was much less when the same measurements were done after removal of the B domain (16). 6).…”