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J. Biol. Chem., Vol. 282, Issue 35, 25453-25463, August 31, 2007

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Structure of Substrate-free Human Insulin-degrading Enzyme (IDE) and Biophysical Analysis of ATP-induced Conformational Switch of IDE^*

Hookang Im¹, Marika Manolopoulou¹, Enrico Malito¹, Yuequan Shen, Ji Zhao, Marie Neant-Fery, Ching-Yu Sun^¶, Stephen C. Meredith^||, Sangram S. Sisodia^¶, Malcolm A. Leissring, and Wei-Jen Tang²

From the Ben-May Department for Cancer Research, the University of Chicago, Chicago, Illinois 60637, the Department of Biochemistry, The Scripps Research Institute, Jupiter, Florida 33458, and the ^¶Department of Neurobiology and ^||Department of Pathology, the University of Chicago, Chicago, Illinois 60637

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid- (A) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that A is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in V_max with only minimal changes to K_m. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

Received for publication, February 22, 2007 , and in revised form, May 22, 2007.

The atomic coordinates and structure factors (code 2jg4 and 2jbu) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM81539, the University of Chicago Diabetes Center pilot and feasibility grants (to W.-J. T.), American Health Assistance Foundation (to E. M.), American Heart Association postdoctoral fellowship (to Y. S.), and Ellison Medical Foundation grant (to M. L.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods and Figs. 1–5.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Tel.: 773-702-4331; Fax: 773-702-4476; E-mail: wtang{at}uchicago.edu.

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