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J. Biol. Chem., Vol. 283, Issue 21, 14893-14900, May 23, 2008

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Structural Basis for Ufm1 Processing by UfSP1^*

Byung Hak Ha¹, Hee-Chul Ahn, Sung Hwan Kang¹, Keiji Tanaka^¶, Chin Ha Chung², and Eunice EunKyeong Kim³

From the Life Sciences Division, Korea Institute of Science and Technology, 39-1 Hawolkok-dong, Sungbuk-gu, Seoul 136-791, Korea, the School of Biological Sciences, Seoul National University, Seoul 151-742, Korea, and the ^¶Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan

Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7Å resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys⁵³, Asp¹⁷⁵, and His¹⁷⁷ that form a catalytic triad, and Tyr⁴¹ that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the "Asp-Pro-His" box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48^USP, seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the K_D value was estimated to be 1.6 µM by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between β3 and 2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1.

Received for publication, October 23, 2007 , and in revised form, February 20, 2008.

The atomic coordinates and structure factors (code 2Z84) 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 grants from the Functional Proteomics Center, the 21st Century Frontier Research and Development Program of the Korea Ministry of Science and Technology and the Korea Institute of Science and Technology Institutional Program Grant (to E. E. K.) and by grants from the Korea Research Foundation and the Korea Science and Engineering Foundation (to C. H. C.). 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.

¹ Recipient of the BK21 fellowship from the Korea Ministry of Education.

² To whom correspondence may be addressed. Tel.: 82-2-880-6693; Fax: 82-2-871-9193; E-mail: chchung{at}snu.ac.kr.

³ To whom correspondence may be addressed. Tel.: 82-2-958-5937; Fax: 82-2-958-5909; E-mail: eunice{at}kist.re.kr.

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