Conformational dynamics of the Escherichia coli maltose transporter

Frances Joan D Alvarez, Purdue University

Abstract

The maltose transporter in Escherichia coli is composed of MalF and MalG, which form a transmembrane pore for passage of maltose, and of MalK2, which binds and hydrolyzes ATP. A wealth of biochemical and structural data strongly supports an alternating access model for the maltose transporter, wherein the maltose binding site in MalFG is alternately exposed to either sides of the membrane, facilitating the translocation of maltose coupled to the hydrolysis of ATP. The inter-conversion of the transporter between the inward and outward facing conformations is initiated by the maltose binding protein (MBP) and reset by ATP hydrolysis. The subunits of the transporter are co-purified and stabilized in detergent micelles, which is a poor mimic of the membrane bilayer, as indicated by the high basal activity of MalFGK2 independent of the stimulation of MBP. Traditionally, reconstitution of MalFGK2 involves incorporation in liposomes, wherein the basal activity of the transporter is abolished and the stimulation of ATPase activity by MBP is remarkably enhanced. However, membrane proteins can insert into liposomes in two different orientations, and surfaces facing the lumen of the vesicle can be inaccessible to ligands or interacting partners. To overcome this limitation, the transporter was reconstituted in bicelles, which are disk-shaped bilayers that form from a predefined mixture of phospholipids and detergents, and in nanodiscs, which are soluble disk-shaped membrane patches that are formed by a phospholipid bilayer encircled by a membrane scaffold protein. The MalFGK2 was shown to recover its MBP-dependent ATP hydrolysis with little to no basal activity in both bicelles and nanodiscs. While both membrane mimics represent alternative reconstitution systems for MalFGK2, reconstitution in nanodiscs is more convenient and efficient. Nanodiscs have also been shown to be a versatile tool suitable for studying the transporter using size exclusion chromatography and electron paramagnetic resonance spectroscopy. The transport activity of MalFGK2 is thought to regulate the transcription of the genes involved in the maltose metabolism, mediated by the interaction of MalK with MalT, which is the activator of the maltose regulon. MalK, associated with MalFG, recruits MalT at the membrane, preventing its binding to DNA. Once maltose transport proceeds, MalK releases MalT, signaling the expression of proteins necessary to degrade maltose. To test this hypothesis, the physical contact between MalT and MalFGK2 was probed by co-elution on nickel affinity resin and size exclusion chromatography. MalFGK2 and MalT exhibited direct interaction in experiments performed in the presence of detergent micelles or using the nanodisc reconstituted transporter. In the alternating access mechanism of membrane transport, the transmembrane domains undergo a series of conformational changes to alternately expose the substrate binding site to each side of the membrane, facilitating transport of the substrate. In an effort to understand the dynamics of the maltose transporter as it alternates between these conformations, spin labels along the transmembrane domains were introduced which can report: 1) on the changes in the mobility on selected sites; and 2) on the relative distances of helices to detect rigid-body motion, bending or twisting motions, using electron paramagnetic resonance spectroscopy. Pairs of spin labels on the cytoplasmic (MalF-391R1/MalG-176R1 and MalF-429R1/MalG217R1) and on the periplasmic (MalF-445R1/MalG-234R1) regions of MalFG were found to move towards or away form each other in a manner consistent with the alternating access model. These spin labels may then be used in the future to obtain dynamic information to clarify the sequence of events in the transport process and to further characterize conformational intermediates in the catalytic cycle. (Abstract shortened by ProQuest.) To investigate the mechanism of stimulation of MBP, site-directed spin labeling electron paramagnetic resonance spectroscopy was used to study the interactions between the C-terminal(C)-lobe of MBP and the transporter. Changes in mobility of nitroxide spin labels attached to MBP or to MalF indicated that the C-lobe of MBP did not bind to the nucleotide-free transporter, where the MalK dimer was in an open conformation. However, the C-lobe was engaged when MalK2 was in a semi-open conformation with MgADP bound or a closed conformation with ATP bound. In detergent solution, MBP stabilized the semi-open MalK2 configuration regardless of the presence of maltose, whereas in reconstituted membrane bilayers, this stabilization required maltose. Similarly, rates of MBP-stimulated ATP hydrolysis were independent of maltose in detergent, but greatly reduced in the absence of maltose in the bilayer. Based on these results, it is suggested that stabilization of the semi-open MalK2 configuration by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

Degree

Ph.D.

Advisors

Davidson, Purdue University.

Subject Area

Chemistry|Biochemistry|Biophysics

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