Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.

Abstract

Maltose transport across the cytoplasmic membrane of Escherichia coli is dependent on the presence of a periplasmic maltose-binding protein (MBP), the product of the malE gene. The products of the malF, maIG, and malK genes form a membrane-associated complex that catalyzes the hydrolysis of ATP to provide energy for the transport event.Previously, mutants were isolated that had gained the ability to grow on maltose in the absence of MBP. After reconstitution of the transport complex into proteoliposomes, measurement of the ATPase activity of wild-type and mutant complexes in the presence and absence of MBP revealed that the wild-type complex hydrolyzed ATP rapidly only when MBP and maltose were both present. In contrast, the mutant complexes have gained the ability to hydrolyze ATP in the absence of maltose and MBP. The basal rate of hydrolysis by the different mutant complexes was directly proportional to the growth rate of that strain on maltose, a result indicating that the constitutive ATP hydrolysis and presumably the resultant cyclic conformational changes of the complex produce maltose transport in the absence of MBP. These results also suggest that ATP hydrolysis is not directly coupled to ligand transport even in wild-type cells and that one important function of MBP is to transmit a transmembrane signal, through the membrane-spnning MaIF and MalG proteins, to the MalK protein on the other side of the membrane, so that ATP hydrolysis can occur.

Characterization of the Adenosine Triphosphatase Activity of the Periplasmic Histidine Permease, a Traffic ATPase (ABC Transporter)

“…This higher value may represent the full hydrolyzing capability of HisQMP 2 since it is comparable to the highest values measured for hisP mutants that have signal-independent activities (32,43). Similar levels of stimulated activity have been reported for the maltose transport complex (reconstituted from solubilized membrane vesicles in PLS): 390 nmol/min/mg of protein (14). Purified HisQMP 2 hydrolyzes ATP with a V max of 2100 nmol/min/mg of protein (Fig.…”

“…If ATP is trapped internally and HisJ is added externally, an accurate measurement of initial rates is very difficult because the internal ATP pool is rapidly exhausted 2 and the accumulated ADP inhibits the reaction (6). Alternatively, trapping HisJ inside the PLS and supplying ATP externally (6,14) presents several drawbacks: the procedure for trapping the receptor is cumbersome, a large amount of receptor protein is required, and a sizable fraction of the complexes must be reproducibly inserted in the inverted orientation; furthermore, because only one or two molecules of receptor can be trapped per PLS vesicle, 3 it would be difficult to study the kinetics of the interaction between the receptor and the complex. Therefore, a permeabilizing procedure that renders ATP and HisJ freely accessible to both sides of PLS is desirable.…”

“…The histidine permease has been reconstituted into PLS that transport histidine when ATP is trapped internally and HisJ is added externally (6,12). As usually assumed in current working models, the liganded receptor sends a signal that initiates ATP hydrolysis and results in subsequent ligand translocation (13,14).…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…The point of substrate release may also vary. Nevertheless, despite such variations, the transition between 'open' and 'closed' NBD dimer, and associated conformational changes, provides a common mechanism for transport ment for binding substrate (histidine or maltose complexed with their respective PBPs) to initiate transport is lost, and, consequently, ATP is hydrolysed continuously in a futile cycle [23,24]. It is of interest that whilst some of these mutants are in the NBDs, others are in the TMDs demonstrating that signals from the TMDs are transmitted to the NBDs to initiate the ATP catalysis cycle.…”

“…The available data suggest that it is binding of ATP to the NBDs which is enhanced, lowering the activation energy for 'closed NBD dimer' formation. First, a mutant E. coli maltose transporter in which the requirement for substrate to stimulate ATP hydrolysis has been lost [24] has a 30-fold increase in NBD affinity for ATP [30]. Secondly, for mammalian P-gp, the transport substrate vinblastine increases the apparent affinity of the NBDs for ATP nearly 60-fold (Linton, Wooding and Higgins, submitted for publication), and fluorescent probes for nucleotide binding have shown that several drugs increase the affinity for ATP [34].…”