Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

Abstract

The ATPase activity of the maltose transporter (MalFGK 2 ) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK 2 are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose-and sucrose-bound sMBP stimulate equal levels of MalFGK 2 ATPase activity. Therefore, the ATPase activity of MalFGK 2 is coupled to translocation of maltose solely by interactions between MalFGK 2 and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the ATP-binding cassette (ABC)2 transporters move various substrates across membranes, with substrate movement coupled to the hydrolysis of ATP. Although the ATPase activity of ABC exporters like P-glycoprotein is generally stimulated by substrate binding, the ATPase activity of ABC importers is activated by a peripheral substrate-binding protein and not the free substrate (for recent reviews see Refs.

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

“…The predominant solution scattering shape of MBP was restored by averaging 10 individual dummy residue models derived from the SAXS data of MBP at a protein concentration of 0.7 mg/ml (data not shown). Interestingly, manual placement of the crystal structure of MBP (PDB code 3HPI, chain A) showed a good fit with the SAXS-derived envelope volume (32). This confirmed that MBP predominantly exists as monomer in solution and the dimeric state of HAMP1-5 MBP was driven intrinsically.…”

The structural basis for MCM2–7 helicase activation by GINS and Cdc45

“…Extra density attributed to MBP on Mcm6 and Mcm3 is colored orange or light blue, respectively; only a portion of the Mcm4 tail is observable in the full-length constructs and is highlighted in green. ( h ) Density corresponding to MBP on Mcm3 (light blue) or Mcm6 (orange), cut out from the CMG density maps and compared to the density map calculated from the MBP crystal structure, PDB entry 3HPI 51 , filtered to 30 Å and showed at 5σ (gray).…”

Molecular mechanism of the Escherichia coli maltose transporter

“…Two laboratories have tested whether direct binding of maltose to MalFGK 2 is required for ATP hydrolysis. Shilton and colleagues designed a MBP mutant that binds both maltose and sucrose [16]. Genetic studies have shown that sucrose is a poor competitor for the maltose-binding site in MalFGK 2 , suggesting that the maltose-binding site in MalFGK 2 does not recognize sucrose [17,18].…”

“…Genetic studies have shown that sucrose is a poor competitor for the maltose-binding site in MalFGK 2 , suggesting that the maltose-binding site in MalFGK 2 does not recognize sucrose [17,18]. However, the mutant MBP stimulated the ATP hydrolysis equally well with maltose or sucrose, indicating that the closed conformation of MBP is sufficient to initiate the transport cycle [16]. Davidson and colleagues substituted an aspartate for glycine in the maltose-binding site to prevent maltose binding to MalFGK2.…”