Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry.
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.
Howard Hughes Medical Institute and Department of Physiology and Microbiology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Adam
A
Weinglass
Julian P
JP
Whitelegge
Kym F
KF
Faull
H Ronald
HR
Kaback
D001120:Arginine; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry*; D018698:Glutamic Acid; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D055672:Static Electricity; D027981:Symporters / Q000737:chemistry*
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D018698:Glutamic Acid; D001120:Arginine