SummaryThe ATP binding cassette (ABC-) transporter mediating the uptake of maltose/maltodextrins in Escherichia coli/Salmonella enterica serovar Typhimurium is one of the best characterized systems and serves as a model for studying the molecular mechanism by which ABC importers exert their functions. The transporter is composed of a periplasmic maltose binding protein (MalE), and a membrane-bound complex (MalFGK 2), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK. We report on the isolation of suppressor mutations within malFG that partially restore transport of a maltose-negative mutant carrying the malK809 allele (MalKQ140K). The mutation affects the conserved LSGGQ motif that is involved in ATP binding. Three out of four suppressor mutations map in periplasmic loops P2 and P1 respectively of MalFG. Cross-linking data revealed proximity of these regions to MalE. In particular, as demonstrated in vitro and in vivo, Gly-13 of substrate-free and substrate-loaded MalE is in close contact to Pro-78 of MalG. These data suggest that MalE is permanently in close contact to MalG-P1 via its N-terminal domain. Together, our results are interpreted in favour of the notion that substrate availability is communicated from MalE to the MalK dimer via extracytoplasmic loops of MalFG, and are discussed with respect to a current transport model.

ATP-binding cassette (ABC) transporters couple the translocation of solutes across membranes to ATP hydrolysis. Crystal structures of the Escherichia coli maltose importer (MalFGK 2 ) in complex with its substrate binding protein (MalE) provided unprecedented insights in the mechanism of substrate translocation, leaving the MalE-transporter interactions still poorly understood. Using pulsed EPR and cross-linking methods we investigated the effects of maltose and MalE on complex formation and correlated motions of the MalK 2 nucleotide-binding domains (NBDs A TP-binding cassette (ABC) systems are found in all kingdoms of life, forming one of the largest protein superfamilies (1-4). ABC transporters comprise two transmembrane domains (TMDs) that form the translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Based on biochemical and structural evidence, all ABC transporters are thought to function by an "alternate-access" mode, with the translocation path shuttling between an inward-facing and outward-facing conformation in response to substrate and ATP binding, the latter causing the NBD dimer to close (5).Canonical ABC importers are subdivided into type I and type II based on structural and biochemical evidence (6) and are dependent on extracellular (or periplasmic) substrate binding proteins (SBPs) (4), which play a crucial role in initial steps of the transport cycle (7-10). SBPs generally consist of two symmetrical lobes that rotate toward each other upon substrate binding (11).The type I maltose transporter of Escherichia coli/Salmonella is probably the best understood ABC transporter to date (12). It is composed of the periplasmic maltose binding protein, MalE, the membrane-integral subunits, MalF and MalG, and the nucleotidebinding subunits (NBDs), MalK 2 . The available crystal structures in the pretranslocation, ATP-, and vanadate-trapped states (13-16) have largely contributed to the understanding of the details of the inward-to outward-facing mechanism. The posthydrolytic state has not yet been crystallized, but data exist proposing this state to have a distinct structure from the other three known crystal snapshots (8, 9). MalE interacts with the transporter throughout the nucleotide cycle (15-18), and the X-ray structures revealed the switch of the binding protein from the liganded (closed) to the substrate-free (open) conformation concomitantly with ATP binding to the NBDs. Despite all these structural insights, the response of the transporter to substrate availability is poorly understood. Furthermore, the mechanism behind the stimulation of the ATPase activity of the transporter by unliganded MalE (10, 19) is still elusive (20,21).Our results show that the apo-and ADP-states of the transporter bind both open and closed MalE, but the complex adopts different periplasmic configurations. The ATP-state of the transporter can bind either closed MalE, inducing its opening and release of substrate to MalF or directly unliganded MalE, which generates a futile cycle. I...

SummaryUnder phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 Ϯ 0.02 mM and 5.1 Ϯ 0.3 mM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC 2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable.

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K1 ) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter.

Enzyme IIA(Glc) (EIIA(Glc)) of the phosphoenolpyruvate phosphotransferase system for the uptake of glucose in Escherichia coli and Salmonella inhibits the maltose ATP-binding cassette transporter (MalE-FGK2) by interaction with the nucleotide-binding and -hydrolyzing subunit MalK, a process termed inducer exclusion. We have investigated binding of EIIA(Glc) to the MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding site I of EIIA(Glc) is contacting the N-terminal subdomain of MalK while the binding site II is relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal structure [ Chen , S. , Oldham , M. L. , Davidson , A. L. , and Chen , J. ( 2013 ) Nature 499 , 364 - 368 ]. Moreover, EIIA(Glc) was found to bind to the MalK dimer regardless of its conformational state. Deletion of the amphipathic N-terminal peptide of EIIA(Glc), which is required for inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter variant and EPR spectroscopy, we demonstrate that EIIA(Glc) arrests the transport cycle by inhibiting the ATP-dependent closure of the MalK dimer.