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Article

Crystal Structure of a Murine Glutathione S-Transferase in Complex with a Glutathione Conjugate of 4-Hydroxynon-2-enal in One Subunit and Glutathione in the Other: Evidence of Signaling across the Dimer Interface^†^,^‡

Bing Xiao,^§ Sharda P. Singh, Bindu Nanduri, Yogesh C. Awasthi, Piotr Zimniak,*^# and Xinhua Ji*^§

ABL-Basic Research Program, NCIFrederick Cancer Research and Development Center, Frederick, Maryland 21702, Departments of Internal Medicine and of Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, and VA John McClellan Memorial Hospital, Little Rock, Arkansas 72205, and Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77555

Biochemistry, 1999, 38 (37), pp 11887–11894

DOI: 10.1021/bi990468i

Publication Date (Web): August 20, 1999

†

This work was supported by NIH Grants ES07804 (P.Z.) and CA27967 (Y.C.A.) and by the National Cancer Institute, DHHS, under contract with ABL (X.J.). The contents of this publication do not necessarily reflect the views or policies of the DHHS, nor does mention of trade names, commercial products, or organizations imply endorsement by the DHHS or the United States Government.

‡

The coordinates have been deposited with the Protein Data Bank (accession code 1b48).

NCI−Frederick Cancer Research and Development Center.

Department of Internal Medicine, University of Arkansas for Medical Sciences.

The University of Texas Medical Branch.

Address correspondence to these authors. X.J.: phone (301) 846-5035; fax (301) 846-6073; e-mail jix@ncifcrf.gov; postal NCI-FCRDC, P.O. Box B, Frederick, MD 21702. P.Z.: phone (501) 257-4843; fax (501) 257-4822; e-mail pxzimniak@life.uams.edu; postal VA Hospital Medical Research (151/LR), 4300 West 7th St., Little Rock, AR 72205.

Department of Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, and VA John McClellan Memorial Hospital.

Abstract

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 Å resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305−310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSHi.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.