Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli

Abstract

The mannitol transporter from Escherichia coli, EII mtl , belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII mtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC mtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.Membrane-embedded transport proteins can be divided in three different classes based on their energy-coupling mechanism (1): (i) primary transport systems, which comprise transporters that use chemical energy or light to actively transport a specific solute over the membrane; (ii) secondary transporters, which are transporters in which the uphill transport of a solute is coupled to the downhill transport of a chemical compound along a gradient; and (iii) group translocation systems, which couple active transport with the chemical modification of the solute. The Enzyme II sugar transporters of the phosphoenolpyruvate-dependent group translocation system (PTS) 2 (2, 3) are the only known transporters belonging to the third class. These transporters phosphorylate their sugar during transport over the membrane. The Escherichia coli PTS transporters specific for glucose, ␤-glucosides, mannitol, and mannose are the best characterized members (4 -9). Enzyme II proteins consist of two cytoplasmic domains, IIA and IIB, involved in phosphoryl group transfer, and a membrane-spanning IIC domain, showing the sugar specificity (10). In some Enzyme II proteins, like the mannose transporter, a second membrane-spanning domain, IID, is present. Depending on its sugar specificity, Enzyme II occurs as separate domains or as fused constructs between two or three domains (11). In EII mtl , the mannitol-specific transporting and phosphorylating enzyme from E. coli, all three domains IIA mtl (12), IIB mtl (13), and IIC mtl (membrane-embedded domain of EII mtl ) are covalently linked (Fig. 1). Mannitol becomes phosphorylated during transport and is released as mannitol-1-phosphate in the cytoplasm (7). The phosphate group originates from phosphoenolpyruvate (PEP) and is transferred to the various EII sugar translocators in the cytoplasmic membrane via two general PTS proteins, EI and HPr (Fig. 1). The phosphoryl transfer reactions from PEP, via EI, HPr, and the A domain to the B domain of Enzyme II are well understood because three-dimensional structures are available for these proteins combined with detailed pre-steady state kinetic data in the case of the glucose transporter, EII glc , from E. coli (14,15). This type of information is not available for the last phosphorylation step from the B domain to the sugar, bound at the C domain. Moreover, no information is available regarding which residues bind the sugar and which residues facilitate the transport of the sugar.For a better understanding of how Enzyme II proteins work, detailed str...

Crystal structure of a phosphorylation-coupled saccharide transporter

“…9a), the C6-OH would be accessible for phosphorylation. This orientation would also place the C6-OH within hydrogen-bonding distance of the conserved residues Glu334 and His250, whose importance for sugar binding and phosphorylation has been demonstrated in an EIIC that transports mannitol 16,17,32–34 . In contrast, the alternate orientation would position the C6-OH in the protein interior (Supplementary Fig.…”

Structural insight into the PTS sugar transporter EIIC

“…Endogenous bound ligand has also been reported for PtsG[68]. The discovery that the mannitol binding site was positioned asymmetrically within the MtlA dimer led to the further hypothesis that the protomers acted in an alternating fashion, with passage of mannitol through one protomer causing it to switch from high affinity to low affinity and consequently driving the other protomer from a low affinity to a high affinity state capable of binding ligand [69]. This differs from what was observed in the bcChbC crystal structure in which the sugar molecule was bound to both protomers in a symmetric fashion.…”

“…There is currently no evidence of cooperativity in the bcChbC dimer; however, given that the cytoplasmic gate appears to be supplied by the opposing protomer, it is possible that this could provide a mechanism for cooperativity, as sugar exiting the cavity of one protomer could be sensed by the other. Furthermore, residues in the N-terminal half of MtlA (the region corresponding from TM1 to TM4 in bcCHBC) were shown to be in close contact with mannitol, the sugar was shown to be located closer to the periplasm, and substantial differences were observed between solvent exposed residues in MtlA and their expected counterparts in the bcChbc structure[69, 70]. Tryptophan phosphorescence spectroscopy indicated that F97 in MtlA (a predicted cytoplasmic loop residue that overlays TM3 in bcChbC) is part of a β-sheet fold[71].…”

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…This interaction seems to occur only with unphosphorylated EIIB Mtl , because first, the Cys-Asp replacement in EIIB Mtl prevented the interaction with MtlR in yeast-two hybrid experiments (146) and second, replacement of the phosphorylatable Cys with a Ser in soluble EIIB Mtl prevented the inhibitory effect on PmtlA expression observed with wild-type EIIB Mtl (71). In contrast to cysteyl-phosphorylated EIIB Mtl , seryl-phosphorylated EIIB Mtl cannot transfer its phosphoryl group to mannitol (148) and, owing to the lowenergy phosphate bond, probably cannot pass it back to EIIB Mtl . As a consequence, P-Ser-EIIB Mtl accumulates in the cytoplasm, and the phosphorylated mutant EIIB Mtl apparently no longer competes with the EIIB Mtl domain of MtlA for binding MtlR and rendering it inactive (71).…”