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. 2004 Aug;5(8):783-8.
doi: 10.1038/sj.embor.7400201. Epub 2004 Jul 16.

Crystal structure of human otubain 2

Max H Nanao  1 , Sergey O Tcherniuk, Jadwiga Chroboczek, Otto Dideberg, Andréa Dessen, Maxim Y Balakirev
Affiliations

Crystal structure of human otubain 2

Max H Nanao et al. EMBO Rep. 2004 Aug.

Abstract

Ubiquitylation, the modification of cellular proteins by the covalent attachment of ubiquitin, is critical for diverse biological processes including cell cycle progression, signal transduction and stress response. This process can be reversed and regulated by a group of proteases called deubiquitylating enzymes (DUBs). Otubains are a recently identified family of DUBs that belong to the ovarian tumour (OTU) superfamily of proteins. Here, we report the first crystal structure of an OTU superfamily protein, otubain 2, at 2.1 A resolution and propose a model for otubain-ubiquitin binding on the basis of other DUB structures. Although otubain 2 is a member of the cysteine protease superfamily of folds, its crystal structure shows a novel fold for DUBs. Moreover, the active-site cleft is sterically occluded by a novel loop conformation resulting in an oxyanion hole, which consists uniquely of backbone amides, rather than the composite backbone/side-chain substructures seen in other DUBs and cysteine proteases. Furthermore, the residues that orient and stabilize the active-site histidine of otubain 2 are different from other cysteine proteases. This reorganization of the active-site topology provides a possible explanation for the low turnover and substrate specificity of the otubains.

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Figures

Figure 1
Figure 1
Structure of otubain 2. Ribbon diagram of otubain 2, coloured from dark blue (N terminus) to red (C terminus). The catalytic residues Cys 51, His 224 and Asn 226 are shown in a stick representation. Selected secondary structural elements are labelled.
Figure 2
Figure 2
Architecture of the otubain activesite. (A) The active-site cysteine, histidine and helix are structurally conserved between otubain (orange) and other cysteine proteases. Cysteine proteases from clans 1 (MEROPS database classification: 1pip, 8pch), 5 (1hav), 12 (1cmx), 19 (1nbf), 28 (1qmy) and 48 (1euv) are shown in green, with active-site residues shown in a stick representation. The spatial locations of the active-site residues T45 and N226 in otubain 2 are novel compared with previous cysteine protease structures (white asterisk on the left). Additionally, the conformation of the loop leading to the active site (44–48) is shifted towards C51 and H224 compared with other cysteine proteases. Also note that the distance between D48 and H224 is too far for a direct interaction. (B) Reorganization of the otubain 2 active site. Otubain is shown in green, UCH in yellow, the UCH Yuh1 in complex with ubiquitin in purple, HAUSP in red and HAUSP in complex with ubiquitin in orange. The ubiquitin chain from the HAUSP–ubiquitin complex is shown in an orange stick representation and labelled ‘Ub'. The side chain of K46 has been omitted for clarity. An oxyanion hole comprised solely of main-chain amides, rather than main-chain/side-chain interactions found in other cysteine proteases, is seen. Also note that between their bound and unbound states, no conformations from UCH/Yuh1 or HAUSP resemble that of the 44–48 loop in otubain 2.
Figure 3
Figure 3
Sequence alignment of otubains. Conserved residues are blocked in black. Colours above the sequence alignment are indicated as follows: Cys 51 (orange), His 224/Asn 226 (blue), the activesite occluding loop Lys 44–Asp 48 (green), Thr 45 (red) and proposed ubiquitin-binding region (pink). The N-terminal extensions of otubain 1 were removed for clarity. Anoph. gam., Anopheles gambiense; Dros. mel., Drosophila melanogaster; Homo. sap., Homo sapiens; Ratt. nor., Rattus norvegicus; Sch. japon., Schizosaccharomyces japonicus.
Figure 4
Figure 4
Ubiquitin interaction and activesite. Colouring is the same as for Fig 3. Proposed ubiquitin-binding residues are shown in purple and are individually labelled.
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