Abstract
In Escherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell. The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that function in the absence of binding protein are thought to be stabilized in an ATPase-active conformation. To probe the conformation of the nucleotide-binding site and to gain an understanding of the nature of the conformational changes that lead to activation, cysteine 40 within the Walker A motif of the MalK subunit was modified by the fluorophore 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Fluorescence differences indicated that residues involved in nucleotide binding were less accessible to aqueous solvent in the binding protein independent transporter than in the wild-type transporter. Similar differences in fluorescence were seen when a vanadate-trapped transition state conformation was compared with the ground state in the wild-type transporter. Our results and recent crystal structures are consistent with a model in which activation of ATPase activity is associated with conformational changes that bring the two MalK subunits closer together, completing the nucleotide-binding sites and burying ATP in the interface.
Highlights
Brane-spanning transport complex consisting of one MalF, one MalG, and two MalK (MalFGK2) protein subunits mediate maltose transport across the cytoplasmic membrane
We have recently demonstrated that maltose-binding protein (MBP) activates the ATPase activity of MalFGK2 by stabilizing the transition state for ATP hydrolysis, and as a consequence becomes tightly bound to the transport complex in the presence of the transition state analogue vanadate [11]
In the WT system, the ATPase activity of the MalFGK2 transporter is stimulated through an interaction with maltose-bound MBP that stabilizes the transition state for hydrolysis [11]
Summary
Bacterial Strains, Plasmids, and Culture Conditions—Strain HN741 (Escherichia coli K-12 argH his rpsL1 malT (Con) malB⌬13 ⌬atpBC ilv::Tn10/FЈ lacIq Tn5) [14] was transformed with the plasmid pMS421. The malKht gene is a variant of malK encoding six histidines at the 5Ј end that are used for purification of the transport complex [23]. For MIANS modification studies, transport complexes were labeled in detergent solution (buffer B) by incubation at 25 °C with either 5 or 20 M MIANS added from a 0.2 mM stock solution in methanol. Complexes that had been reconstituted into proteoliposome vesicles by dilution [6] were labeled in buffer containing 20 mM NaPi (pH 8) and 10 mM MgCl2 at 37 °C [26]. Assays of ATP Hydrolysis—ATP hydrolysis activity of detergentsoluble and reconstituted transport complexes was measured using [␥-32P]ATP (Amersham Pharmacia Biotech) as described previously [12, 27]. Values are the mean Ϯ S.D. of triplicate determinations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
