1. Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
2. Department of Biological Information, and,
3. Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan
4. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8501, Japan
5. Department of Biophysics and Biochemistry, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan
6. Present address: Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Correspondence to: Shuya Fukai^1,4 Correspondence and requests for materials should be addressed to S.F. (Email: fukai@iam.u-tokyo.ac.jp).

Abstract

Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn²⁺-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 Å and 1.6 Å resolutions, respectively. The AMSH-LP DUB domain consists of a Zn²⁺-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.

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