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J. Biol. Chem., Vol. 279, Issue 40, 41858-41865, October 1, 2004

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Monitoring Conformational Rearrangements in the Substrate-binding Site of a Membrane Transport Protein by Mass Spectrometry^*

Adam Weinglass, Julian P. Whitelegge, Kym F. Faull, and H. Ronald Kaback¶

From the Howard Hughes Medical Institute and Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, the Pasarow Mass Spectrometry Laboratory and Departments of Psychiatry and Behavioral Sciences, Neuropsychiatric Institute and Departments of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095-1662

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 (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.

Received for publication, July 7, 2004 , and in revised form, July 19, 2004.

^* This work was supported by National Institutes of Health Grant DK51131:09 (to H. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: HHMI/UCLA, 5-748 MacDonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623. E-mail: RonaldK{at}HHMI.UCLA.edu.

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