scholarly journals The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle

2008 ◽  
Vol 191 (3) ◽  
pp. 754-761 ◽  
Author(s):  
Martin L. Daus ◽  
Mathias Grote ◽  
Erwin Schneider
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Cross Linking ◽  
Atp Binding ◽  
Cross Link ◽  
Atp Binding Cassette Transporter ◽  
Serovar Typhimurium ◽  
Atp Binding Cassette

ABSTRACT We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK2) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models.

scholarly journals Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

2017 ◽  
Vol 19 (14) ◽  
pp. 9366-9373 ◽  
Author(s):  
Jingwei Weng ◽  
Shuo Gu ◽  
Xin Gao ◽  
Xuhui Huang ◽  
Wenning Wang
Keyword(s):  
Abc Transporter ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Atp Binding ◽  
Type I ◽  
Closed Conformation ◽  
Atp Binding Cassette Transporter ◽  
Maltose Transporter ◽  
Atp Binding Cassette

Maltose transporter MalFGK2is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily.

Periplasmic Loop P2 of the MalF Subunit of the Maltose ATP Binding Cassette Transporter Is Sufficient To Bind the Maltose Binding Protein MalE†

Biochemistry ◽  
2009 ◽  
Vol 48 (10) ◽  
pp. 2216-2225 ◽  
Author(s):  
Tomas Jacso ◽  
Mathias Grote ◽  
Martin L. Daus ◽  
Peter Schmieder ◽  
Sandro Keller ◽  
...  
Keyword(s):  
Binding Protein ◽  
Maltose Binding Protein ◽  
Atp Binding ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette

scholarly journals Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants

1998 ◽  
Vol 180 (9) ◽  
pp. 2507-2514 ◽  
Author(s):  
Bryn D. Nelson ◽  
Beth Traxler
Keyword(s):  
Membrane Proteins ◽  
Cytoplasmic Membrane ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Integral Membrane Proteins ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Transport Complex ◽  
Atp Binding Cassette

ABSTRACT The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2 assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2 complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.

scholarly journals Evidence from Mutational Analysis for a Single Transmembrane Substrate Binding Site in the Histidine ATP-Binding Cassette Transporter ofSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Salmonella Enterica ◽  
Substrate Binding ◽  
Atp Binding ◽  
Substrate Molecule ◽  
Content Type ◽  
Charged Residues ◽  
Serovar Typhimurium ◽  
Atp Binding Cassette

ABSTRACTThe histidine ATP-binding cassette (ABC) transporter ofSalmonella entericaserovar Typhimurium is among the best-studied type I ABC import systems. The transporter consists of two transmembrane subunits, HisQ and HisM, and a homodimer of the nucleotide-binding subunit, HisP. Substrates are delivered by two periplasmic solute binding proteins, HisJ and LAO, with preferences for histidine and for lysine, arginine, and ornithine, respectively. A homology model was built by using the arginine-bound crystal structure of the closely related Art(QN)2transporter ofThermoanaerobacter tengcongensisas the template. In the homodimeric Art(QN)2, one substrate molecule is bound to each of the ArtQ subunits, whereas the structural model and sequence alignments predict only one substrate molecule in contact with HisM. To address the question whether one or two binding sites exist in heterodimeric HisQM, we have studied the functional consequences of mutations by monitoring (i) the complementation of growth ond-histidine of auxotrophic tester strains, (ii) the growth of tester strains on arginine as a nitrogen source, and (iii) ATPase activity of purified variants in a lipid environment. Our results demonstrate that two negatively charged residues, namely, HisM-E166 and HisQ-D61, are indispensable for function. Furthermore, the complete reconstruction of an ArtQ-like binding site in HisQ resulted in an inactive transporter. Likewise, switching the positions of both negatively charged residues between HisQ and HisM caused transport-deficient phenotypes. Thus, we propose that one substrate molecule is primarily liganded by residues of HisM while HisQ-D61 forms a crucial salt bridge with the α-amino group of the substrate.IMPORTANCECanonical ATP-binding cassette (ABC) importers are major players in the translocation of numerous nutrients, vitamins, and growth factors to the cytoplasm of prokaryotes. Moreover, some ABC importers have been identified as virulence factors in bacterial pathogenesis. Thus, a full understanding of their mode of action is considered a prerequisite, among others, for the development of novel antibacterial drugs. However, mainly owing to the lack of structural information, the knowledge of the chemical nature and number of substrate binding sites formed by the transmembrane subunits of ABC importers is scarce. Here, we provide evidence from mutational analyses that, in contrast to homologous homodimeric systems, the heterodimeric histidine transporter ofSalmonella entericaserovar Typhimurium is liganding only one substrate molecule between its transmembrane subunits, HisM and HisQ.

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