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Members of the Amt family of channels mediate the transport of ammonium. The form of ammonium, NH3 or NH4(+), carried by these proteins remains controversial, and the mechanism by which they select against K(+) ions is unclear. We describe here a set of Escherichia coli AmtB proteins carrying mutations at the conserved twin-histidine site within the conduction pore that have altered substrate specificity and now transport K(+). Subsequent work established that AmtB-mediated K(+) uptake occurred against a concentration gradient and was membrane potential-dependent. These findings indicate that the twin-histidine element serves as a filter to prevent K(+) conduction and strongly support the notion that Amt proteins transport cations (NH4(+) or, in mutant proteins, K(+)) rather than NH3 gas molecules through their conduction pores.

In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of sugar 6-phosphate by exchange with internal inorganic phosphate (Pi). The substrate specificity of UhpT is regulated at least in part by constituents of an Asp388-Lys391 intrahelical salt bridge, and mutations that remove one but not both of these residues alter UhpT preference for organophosphate substrates. Using site-directed mutagenesis, we examined the role played by these two positions in the selection of the oxyanion countersubstrate. We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO4 as well as Pi. These oxyanions share similar structures but differ significantly in the presence of a proton(s) on Pi. Our findings therefore lead us to suggest that the Asp388-Lys391 ion pair acts normally as a filter that prevents substrates lacking a proton that can be donated from occupying the UhpT active site.

The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette superfamily, mediates the high affinity uptake of maltose at the expense of ATP. The membrane-associated transporter consists of two transmembrane subunits, MalF and MalG, and two copies of the cytoplasmic ATP-binding cassette subunit, MalK. Maltose-binding protein (MBP), a soluble periplasmic protein, delivers maltose to the MalFGK(2) transporter and stimulates hydrolysis by the transporter. Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK(2) and conformational changes in MBP as it interacts with MalFGK(2). Cysteine residues and spin labels have been introduced into the two lobes of MBP so that spin-spin interaction will report on ligand-induced closure of the protein (Hall, J. A., Thorgeirsson, T. E., Liu, J., Shin, Y. K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614). At least two different modes of interaction between MBP and MalFGK(2) were detected. Binding of MBP to MalFGK(2) in the absence of ATP resulted in a decrease in motion of spin label at position 41 in the C-terminal domain of MBP. In a vanadate-trapped transition state intermediate, all free MBP became tightly bound to MalFGK(2), spin label in both lobes became completely immobilized, and spin-spin interactions were lost, suggesting that MBP was in an open conformation. Binding of non-hydrolyzable MgATP analogs or ATP in the absence of Mg is sufficient to stabilize a complex of open MBP and MalFGK(2). Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P(i)-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys(391), a member of an intrahelical salt bridge (Asp(388)/Lys(391)) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an alpha-helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.

Ligands that are transported by the maltose transport system of Escherichia coli must first bind to the periplasmic maltose-binding protein (MBP). However, binding of a ligand does not always lead to its transport. As reported earlier, reduced or oxidized maltodextrins bind tightly to MBP but are not transported; some mutant MBPs, such as MalE254, bind maltodextrins tightly but cannot produce their transport. In this study, UV differential spectroscopy and fluorescence emission spectroscopy were used to study the modes by which various ligands bind to MBP. Maltose binding produced a red shift in the fluorescence emission spectrum of wild type MBP and a sharp hypochromatic trend below 265 nm in its UV spectrum (R mode (for red)). On the other hand, binding of reduced, oxidized, or cyclic maltodextrins produced a pronounced blue shift in the fluorescence emission spectrum of wild type MBP and a peak at about 250 nm in its UV difference spectrum (B mode (for blue). Binding of reducing maltodextrins to wild type MBP produced spectral changes that seemed to be a mixture of predominantly R mode binding and some B mode binding, whereas their binding to mutant MBP MalE254 produced changes indicative of pure B mode binding. Thus, the ligands that are bound exclusively via the B mode to either the wild type or MalE254 MBP are not transported.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

In the preceding two papers (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609; Hall, J. A., Thorgeirson, T. E., Liu, J., Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614), we showed that ligands that bind to the Escherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the cytoplasm. Here, we examine various combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells. Closed forms of wild type MBP, complexed with maltose or maltodextrins, interacted with wild type transporter complex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2360-2364). In contrast, open forms of liganded MBPs, such as the complex containing wild type MBP and reduced, oxidized, or cyclic maltodextrins or the complex containing the mutant MBP MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolysis, suggesting that the proper interaction between the ligand-MBP complex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter containing MalG511 was used, the already significant basal level of ATP hydrolysis was further stimulated not only by ligand MBPs in the closed form but also by those in the open form (except that containing beta-cyclodextrin), data suggesting that the mutant transporter does not always require the closed MBP complex presumably because of its exceptionally strong affinity to MBP, described earlier (Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992) Mol. Microbiol. 6, 2033-2040). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow by using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process.