Maltose-binding Protein Is Open in the Catalytic Transition State for ATP Hydrolysis during Maltose Transport

Mariana I Austermuhle,Jason A Hall,Candice S Klug,Amy L Davidson

doi:10.1074/jbc.m403508200

Abstract

The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette superfamily, mediates the high affinity uptake of maltose at the expense of ATP. The membrane-associated transporter consists of two transmembrane subunits, MalF and MalG, and two copies of the cytoplasmic ATP-binding cassette subunit, MalK. Maltose-binding protein (MBP), a soluble periplasmic protein, delivers maltose to the MalFGK(2) transporter and stimulates hydrolysis by the transporter. Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK(2) and conformational changes in MBP as it interacts with MalFGK(2). Cysteine residues and spin labels have been introduced into the two lobes of MBP so that spin-spin interaction will report on ligand-induced closure of the protein (Hall, J. A., Thorgeirsson, T. E., Liu, J., Shin, Y. K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614). At least two different modes of interaction between MBP and MalFGK(2) were detected. Binding of MBP to MalFGK(2) in the absence of ATP resulted in a decrease in motion of spin label at position 41 in the C-terminal domain of MBP. In a vanadate-trapped transition state intermediate, all free MBP became tightly bound to MalFGK(2), spin label in both lobes became completely immobilized, and spin-spin interactions were lost, suggesting that MBP was in an open conformation. Binding of non-hydrolyzable MgATP analogs or ATP in the absence of Mg is sufficient to stabilize a complex of open MBP and MalFGK(2). Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.

Highlights

The class of proteins termed ATP-binding cassette (ABC)1 is one of the largest found in nature
We propose that maltose-binding protein (MBP) stimulates the ATPase activity of the transporter by stabilizing the transition state and that attainment of the transition state is coupled to the loss of high affinity for maltose, thereby coupling maltose transport to ATP hydrolysis [13]
ATP-mediated association of the MalK subunits is accompanied by a rearrangement in the membrane-spanning domains (MSDs) that exposes a low affinity maltose-binding site in the transmembrane region to the periplasm, and simultaneously, MBP opens to release maltose into the transporter

Summary

Conformational Changes in MBP during Maltose Transport

Low only the monitoring of global changes in protein structure, SDSL allows the direct probing of the local environment, structure, and proximity of individual residues. SDSL typically involves introducing a placed cysteine residue within a protein followed by a reaction with a sulfhydryl-specific nitroxide spin label. Conformational changes in protein structure can be observed through both solvent accessibility measurements and motional changes of the introduced spin label side chain. Conformational changes due to substrate binding; membrane binding; secondary, tertiary, and protein-protein interactions; denaturation; and other perturbations are all evident in the EPR spectra and can give key information on structural changes at specific sites. The observation of alterations in distance between sites upon ligand binding or protein-protein interaction is a powerful approach to characterizing conformational changes [18, 19]. We use SDSL and EPR to study the dynamics of MBP as it interacts with the maltose transporter during nucleotide binding and ATP hydrolysis.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION