Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

Scheek³

et al. 1998

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

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Meijberg

Scheek³

et al. 1998

“…, carrying the various plasmids, and procedures to overexpress the mutant proteins were identical to those described for wild type EII mtl (13). Inside-out (ISO) membrane vesicles containing the mutant proteins were obtained as described (14).…”

Section: Isolation Of Iso Membrane Vesicles and Membrane Solubilization-mentioning

confidence: 99%

Mapping of the Dimer Interface of the Escherichia coli Mannitol Permease by Cysteine Cross-linking

Montfort

Wind

et al. 2002

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline) 3 -catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning ␣-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys 73 ). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys 134 ), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys 134 becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for ␣-helix packing in the mannitol permease.In bacteria, the phosphoenolpyruvate-dependent phosphotransferase system is involved in the uptake and concomitant phosphorylation of a wide variety of carbohydrates (1). The system consists of two general components EI and HPr and several carbohydrate-specific enzymes II (EII). In a cascade of phosphorylation reactions, the phosphoryl group is transferred from the energy donor phosphoenolpyruvate via EI and HPr to EII, as is illustrated in Fig. 1 for the mannitol-specific phosphotransferase system (reviewed in Ref. 2). All EII's have a similar architecture and consist of the cytoplasmic A and B domains and a membrane-embedded C domain. In the mannitol-specific EII (EII mtl ) 1 of Escherichia coli the three domains are covalently linked. The C domain transports mannitol into the cell and the A and B domains transfer the phosphoryl group from HPr to mannitol. The imported mannitol is phosphorylated by B while being bound by C. The association state of EII mtl is most likely the dimer with contacts between two subunits being provided by the C domain. The evidence for this oligomeric state is manifold, but the strongest indications are from in vitro and in vivo complementation studies. The formation of heterodimers from inactive subunits with defects in different domains restores the activity of EII mtl (3-5). Moreover, subunits of EII mtl could be covalently cross-linked to a dimer with bifunctional sulfhydryl-specific reagents, lysine-specific cross-linkers, or by oxidative disulfide bond formation (6 -9). The 5-Å projection structure of the C domain in two-dimensional crysta...

“…c , ⌬(gutRЈMDBA-recA)) carrying the various plasmids, and procedures to overexpress the mutant proteins were identical to those described for wild-type EII mtl (20). Inside-out (ISO) membrane vesicles containing the mutant proteins were obtained as described (21).…”

Section: Generation Of Iso Vesicles and Purification Of Sscs Sscs-s1mentioning

confidence: 99%

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

Montfort

Duurkens

et al. 2001

Part of the dimer and B/C domain interface of theThe uptake and concomitant phosphorylation of a wide variety of carbohydrates into bacterial cells is, in many cases, accomplished by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (1). In a cascade of phosphorylation reactions (Fig. 1) Mannitol in the periplasm is bound by the C domain, transported into the cell via C and, while bound at the cytoplasmic site of C, phosphorylated by the B domain. EII mtl is most likely a dimeric protein and the subunit interactions occur in the C domain (3-8).Domain interactions and in particular the B/C domain interface play an important role in the catalytic cycle of EII mtl . The energy coupling mechanism involves conformational interaction between the B and C domain. The evidence for this notion is manifold. 1) Phosphorylation of the B domain increases the rate of transport 2-3 orders of magnitude (9, 10). 2) Modification or mutagenesis of the phosphorylation site in the B domain as well as removal of the cytoplasmic domains changes the mannitol binding kinetics of the C domain (11,12). 3) Timeresolved fluorescence and phosphorescence spectroscopy showed that, upon phosphorylation of the B domain, Trp 109 in the C domain becomes immobilized whereas Trp 30 in the C domain becomes more flexible (13,14). 4) Differential scanning calorimetry showed that the thermal stability of the C domain is higher in the presence of the B domain (15). 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). Close proximity of the B and C domain has been suggested for another PTS transporter, that is the BglF system of E. coli. 3To date, there is no structural information about the B/C domain or dimer interface of EII mtl or any other EII. The topological model of the C domain predicts 6 membrane-span-