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J. Biol. Chem., Vol. 279, Issue 9, 8140-8148, February 27, 2004

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The Catalytic Domain of Escherichia coli Lon Protease Has a Unique Fold and a Ser-Lys Dyad in the Active Site^*

Istvan Botos, Edward E. Melnikov, Scott Cherry, Joseph E. Tropea, Anna G. Khalatova, Fatima Rasulova¶, Zbigniew Dauter, Michael R. Maurizi¶, Tatyana V. Rotanova, Alexander Wlodawer, and Alla Gustchina||

From the Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, and the ^¶Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland 20892

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-Å resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of 18 Å. Within the active site, the proximity of Lys⁷²² to the side chain of the mutated Ala⁶⁷⁹ and the absence of other potential catalytic side chains establish that Lon employs a Ser⁶⁷⁹-Lys⁷²² dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.

Received for publication, November 7, 2003 , and in revised form, November 28, 2003.

The atomic coordinates and structure factors (codes 1rr9 and 1rre) 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 in part by Russian Foundation for Basic Research Grant 02-04-48481 (to T. V. R.) and by United States Civilian Research and Development Foundation Grant RB1–2505-MO-03 (to T. V. R. and A. W.). 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: National Cancer Institute, MCL, Bldg. 539, Rm. 143, Frederick, MD 21702-1201. Tel.: 301-846-5338; Fax: 301-846-6128, E-mail: alla{at}ncifcrf.gov.

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