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.
We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK(2)) 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) ( approximately 5 A) 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 approximately 10 A to approximately 5 A upon binding of ATP (or maltose, with a less pronounced result) and was reset to approximately 10 A after hydrolysis of one ATP. A cross-link ( approximately 25 A) 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.
Daus
Martin L
ML
Institut für Biologie, AG Bakterienphysiologie, Humboldt-Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany.
Grote
Mathias
M
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C468279:MalFGK(2) protein, Salmonella typhimurium; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D000255:Adenosine Triphosphate