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

Mariana I. Austermuhle,J. Hall,C. Klug,A. Davidson

Published 2004 in Journal of Biological Chemistry

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 MalFGK2 transporter and stimulates hydrolysis by the transporter. Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK2 and conformational changes in MBP as it interacts with MalFGK2. 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 MalFGK2 were detected. Binding of MBP to MalFGK2 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 MalFGK2, 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 MalFGK2. Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.

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