We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK 2 ) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model. ATP-binding cassette (ABC)3 transporters are involved in the uptake or export of an enormous variety of substances ranging from small ions to large polypeptides at the expense of ATP. They are found in all organisms from bacteria to humans, and dysfunction is often associated with disease in humans, such as cystic fibrosis, adrenoleukodystrophy, or Stargardt's macular dystrophy (1). ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide binding (ABC) domains (NBDs) that hydrolyze ATP. In fact, in most prokaryote importers, TMDs and NBDs are expressed as separate protein subunits, whereas in most export systems of both prokaryotes and eukaryotes, they are usually fused into a single polypeptide chain (2).The ABC domains are characterized by a set of Walker A and B motifs that are involved in nucleotide binding and by the unique "LSGGQ" signature sequence (3). The crystal structures of several mostly prokaryotic NBDs have been reported that largely agree on the overall fold (reviewed in Refs. 4 -6). Accordingly, the cassette can be divided into a RecA-like subdomain encompassing both Walker m...

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants.

We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys 262 of MalF and Arg 73 of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln 140 appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATPbinding cassette import system upon binding of ATP. ATP-binding cassette (ABC)3 proteins exist in all living organisms and form one of the largest superfamilies. They are integral to almost every biological process and physiological system. Most are involved in the uptake or export of an enormous variety of substances across cell membranes, from small ions to large polypeptides. Many of these proteins are of considerable medical importance. Mutations in several "ABC genes" lead to genetic diseases, such as cystic fibrosis, Tangier disease, and adrenoleukodystrophy, or confer resistance to antibiotics and chemotherapeutic agents (1).ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide-binding (ABC) domains (NBDs) that hydrolyze ATP. In prokaryotes, these domains are mostly expressed as separate protein subunits, whereas in eukaryotes, especially in mammalian cells, they are usually fused into a single polypeptide chain.The ABC domains are characterized by a set of Walker A and B motifs that are involved in nucleotide binding and by the unique LSGGQ signature sequence (2). To date, the crystal structures of several mostly prokaryotic ABC domains have been reported (reviewed i...

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.

Edited by Paul E. Fraser Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrP C . The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrP Sc from PrP C using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrP Sc with reduced sialylation levels using the same method but with partially desialylated PrP C as a substrate (dsPMCAb (22,23). Sialylated glycans play an essential role in a broad range of cellular functions, but are especially important in immunity (24). Terminal sialic acid residues on the surface of mammalian cells act as a part of a "self-associated molecular pattern," providing molecular cues to the immune system for discriminating between "self," "altered self," or "non-self" (25, 26). Glycoproteins or glycolipids that lack sialic acids at terminal positions serve as a "pathogen-associated molecular pattern" used by mammalian immune systems to recognize pathogens or asialoglycoproteins that need to be removed (25). Bearing in mind that sialylation serves as a molecular marker of self versus nonself, we proposed a hypothesis that sialylation determines the fate of prions in an organism (27)