As a 42-kDa multi-domain protein, AT3 consists of an N- terminal compact Josephin domain, two UIM motifs, a polyQ stretch, and a short variable tail (Fig. 1A) [23]. There is another UIM (UIM3) at the C-terminal region, which was only identified in a splice variant [24]. The Josephin domain itself can bind Ub and has deubiquitinating activity, which functions in regulating P97/VCP-associated retrotranslocation [25]. The binding of Ub to AT3 is also mediated by its UIMs, and single-point mutation of the conserved serine residues in the UIM motifs can eliminate its binding to ubiquitinated proteins [11]. Hence, the UIM regions have been proposed to recruit polyubiquitinated substrates to position the polyUb chains adjacent to the catalytic site, and/or to allow the enzyme to act in trimming polyUb chains in a distal to proximal direction [26]. It is reasonable to speculate that multiple copies of UIM would be conducive to this function. The UIM sequences (,20 residues) of AT3 are similar to those found in many other proteins, such as S5a, Vps27p, AT3, and RAP80 (Fig. 1B) [15]. These proteins each contain two tandem UIM motifs; however, the linker regions between these UIM motifs are largely different. The UIMs of S5a and Vps27p are interrupted by a long linker of 20,50 residues, whereas the two UIM motifs of AT3 are juxtapositional to each other in sequence with the shortest linker. This short linker may provide a possibility that the two UIM motifs bind Ub cooperatively. The Ub binding property of AT3 has linked its normal biological function to protein surveillance pathway [12]. Accu- mulation of evidence suggests that the Ub-proteasome pathway also participates in the pathogenesis of the diseases [27,28,29]. To clarify the specific recognition between tandem UIMs of AT3 (AT3-UIM12) and Ub chains or ubiquitinated substrates that may AT3-UIM/Ub Interaction be of functional significance, we determined the solution structures of AT3-UIM12 in free and bound forms, analyzed its binding properties with Ub or Ub chains by NMR, isothermal titration calorimetry (ITC) and other biochemical techniques. The importance of the linker region in the UIM motifs was clearly addressed and a possible interaction mode between the two tandem UIMs of AT3 and Ub or Ub chains was also proposed and discussed in detail. Results Solution Structures of the Free and Ub-Bound Forms of Tandem AT3-UIM12 To obtain the binding properties of the tandem UIM motifs with Ub, we firstly assigned the chemical shifts (Supplemental Fig. S1A) and NOEs of AT3-UIM12, and solved its solution structure by heteronuclear multidimensional NMR techniques. A summary of the NMR-derived conformational restraints for structure calcula- tion and the structure model statistics is presented in Supplemental Table S1. Similar to the structures of other tandem UIMs [16,18], the overall structure of AT3-UIM12 is poorly defined due to relative flexibility of the linker, but the two individual UIM regions are well structured (Fig. 2). We depict an ensemble of 10 lowest-energy NMR structures superimposed on the polypeptide backbones of helix-1 (Fig. 2A) and helix-2 (Fig. 2B), respectively. As expected from the sequence, the structure of AT3-UIM12 is comprised of two a- helices and a flexible linker, 239EIDMED244. Due to the absence of long-range NOEs (Supplemental Table S1), the tertiary interactions between the two helices are also absent; hence, the orientation of the two helices is less restricted. Thus, the short linker between the two Figure 1. Domain Architecture of AT3 Protein and Sequence Alignments of the UIM Motifs. A, Domain architecture of AT3 protein. B, Multiple sequence alignments of the UIM motifs from AT3, S5a, Vps27 and RAP80. Note that the length of the linker region between the two tandem UIM motifs varies in these four proteins. The linker in AT3 is the shortest (6 residues), and then in RAP80 (,11 residues), whereas those in S5a and Vps27p are longer with 50 and 23 residues, respectively. The sequences were aligned using ClustalW and represented using ESPript. C, Sequences of the linker regions for wild-type AT3-UIM12 and its mutants. GS-Sub, substitution with GSSGGS sequence; GS-Ins, Insertion of GSSGGS; RAP80-Linker, replacement with the linker sequence of RAP80. doi:10.1371/journal.pone.0013202.g001 PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13202