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J. Biol. Chem., Vol. 279, Issue 12, 11432-11443, March 19, 2004

This Article Full Text Full Text (PDF) All Versions of this Article: 279/12/11432    most recent M310903200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kroczynska, B. Articles by Blond, S. Y. Search for Related Content PubMed PubMed Citation Articles by Kroczynska, B. Articles by Blond, S. Y. Social Bookmarking                      What's this?

The SANT2 Domain of the Murine Tumor Cell DnaJ-like Protein 1 Human Homologue Interacts with ₁-Antichymotrypsin and Kinetically Interferes with Its Serpin Inhibitory Activity^*

Barbara Kroczynska, Christina M. Evangelista, Shalaka S. Samant, Ebrahim C. Elguindi, and Sylvie Y. Blond

From the Center for Pharmaceutical Biotechnology, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois 60607-7173

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620–19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with ₁-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nM. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

Received for publication, October 2, 2003 , and in revised form, December 8, 2003.

^* This work was supported by National Institutes of Health Grant NIHGM 58107 (to S. Y. B.) and the Talaat Basha Family Foundation (to E. C. E.). 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: Center for Pharmaceutical Biotechnology (M/C 870), University of Illinois, Molecular Biology Research Bldg., 900 South Ashland Ave., Chicago, IL 60607-7173. Tel.: 312-996-5416; Fax: 312-413-9303; E-mail: blond{at}uic.edu.

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