Abstract
Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding.
Highlights
The major facilitator superfamily (MFS)1 is one of the largest families of membrane transport proteins found in archaeal, bacterial, and eukaryotic cell membranes [1] and contains over 1000 members, some of which are clinically relevant
LacY is selective for disaccharides containing a D-galactopyranosyl ring as well as D-galactose (10 –12) but does not interact with D-glucopyranosides or D-glucose [12,13,14]
Using the x-ray structure of the inward facing conformation with substrate bound as a structural foundation, mass spectral techniques have been exploited to gain detailed insights into local alterations in structure induced by substrate binding
Summary
Materials—TDG, NPGal, p-nitrophenyl-␣-D-glucopyranoside (NPGlc), diisopropylcarbodiimide (DiPC), and BD were purchased from Sigma. n-Dodecyl--D-maltopyranoside (DDM) was purchased from Calbiochem. Construction of LacY Mutants—Two-step PCR mutagenesis of the genes encoding wild-type and C154G LacY (in plasmid pT7-5 bearing a His tag) generated products containing the double mutation R135M/ R142S. These PCR products were subcloned back into wild-type LacY as PstI/KpnI fragments. B, time courses are shown for active transport by E. coli T184 (lacZϪYϪ) expressing wild-type LacY [1], no LacY (2, pT7-5 with no lacY insert), mutant C154G [7], mutant R135M/R142S/C154G [8], mutant E126A/R135M/R142S/C154G [9], E269A/R135M/ R142S/C154G [10], and E126A/E269A/R135M/R142S/C154G [11]. Aliquots (50 l) of cell suspensions containing 35 g of protein in 100 mM potassium Pi (pH 7.5), 10 mM MgSO4 were assayed at 0.4 mM final external concentration of lactose as described under “Experimental Procedures.”
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