Structural Transformation of the Tandem Ubiquitin- Interacting Motifs in Ataxin-3 and Their Cooperative Interactions with Ubiquitin Chains Ai-Xin Song 1 , Chen-Jie Zhou 1 , Yu Peng 2 , Xue-Chao Gao1, Zi-Ren Zhou 1 , Qing-Shan Fu1, Jing Hong 2 , Dong-Hai Lin2*, Hong-Yu Hu 1 * 1 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, 2 Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Abstract The ubiquitin-interacting motif (UIM) is a short peptide with dual function of binding ubiquitin (Ub) and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12) both in free and Ub- bound forms. The solution structure of free AT3-UIM12 consists of two a-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates. Citation: Song A-X, Zhou C-J, Peng Y, Gao X-C, Zhou Z-R, et al. (2010) Structural Transformation of the Tandem Ubiquitin-Interacting Motifs in Ataxin-3 and Their Cooperative Interactions with Ubiquitin Chains. PLoS ONE 5(10): e13202. doi:10.1371/journal.pone.0013202 Editor: Vladimir N. Uversky, Indiana University, United States of America Received July 14, 2010; Accepted September 3, 2010; Published October 7, 2010 Copyright: 2010 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the grants from the National Natural Science Foundation of China (30600103, 30670431 and 30870485), the Knowledge Innovation Program of Shanghai Institutes for Biological Sciences (2008KIP201, CAS), and the National Basic Research Program of China (2006CB806508, 2006CB910305 and 2007CB914304). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hyhu@sibs.ac.cn (H-YH); dhlin@mail.shcnc.ac.cn (D-HL) Introduction Ubiquitin (Ub)-interacting motif (UIM) is a highly conserved short peptide sequence found in a number of different proteins and can bind to mono- or poly-ubiquitin (monoUb or polyUb) [1,2]. It was originally identified from the S5a subunit of the 19S proteasome [3], where the UIM interacts with the ubiquitinated proteins or polyUb chains, and facilitates degradation of the selected proteins by proteasome. UIM motifs are also prevalent in the proteins that function in the pathways of endocytosis and vacuolar protein sorting, like Epsins [4], Eps15 [5,6] and Hrs (yeast Vps27p) [7,8], and in the DNA repair pathway, like RAP80 [9]. Ataxin-3 (AT3) is a polyglutamine (polyQ)-containing protein that is related to one kind of the polyQ diseases, spinocerebellar ataxia 3 or Machado-Joseph disease (SCA3/MJD) [10]. It contains two or three conserved UIM motifs around the polyQ tract in sequence that are crucial for binding Ub and promoting ubiquitination of substrates [11,12]. The conserved residues of UIMs, especially the hydrophobic residues, play important roles in Ub recognition. Similar to other known Ub-binding domains, such as Ub-associated domain (UBA), UIM mainly interacts with the Leu8-Ile44-Val70 hydro- phobic patch on Ub molecule [13,14]. The three-dimensional structures of UIM motifs in several proteins have also been solved [15,16,17,18]. Typically, each UIM motif adopts a single a-helix structure in solution, whereas UIM2 from Vps27p forms a four- helix bundle as an oligomer when crystallized [16] and the two UIMs of RAP80 merge into a long helix when bound with K63- linked diUb [19]. Although UIM is a single-helix motif, it displays various features and diverse functions when it binds to Ub. For instance, the double-sided UIM in Hrs protein can bind two Ub molecules simultaneously on both sides [20]. The sequence of the double- sided UIM, characterized by the overlay of two UIMs (a special form of tandem UIMs), has been proposed as a strategy for increasing its binding efficiency. Another example is MIU (motif interacting with Ub) or IUIM (inverted UIM), which is identified through studies of the Rabex-5 GTPase exchange factor [21], the mammalian ortholog of yeast Vps9p. The helix of Rabex-5 binds across the Ile44 surface of Ub in the same position as does a canonical UIM helix, but with the opposite polarity. This interaction represents another interesting variation in the UIM recognition motif. Notably, many proteins contain multiple copies of the UIM motif connected by linkers with different lengths. The repeat of UIM sequence suggests the presence of possible cooperation in the interactions with Ub chains or ubiquitinated substrates, as in the case of an endocytic protein Cue that binds with Ub [22]. However, the previous studies on S5a and Vps27p proteins did not reveal any cooperative effect, even though both of them contain tandem UIMs [16,18]. Instead, their individual UIMs bind Ub independently and non- cooperatively. PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13202