AT3-UIM/Ub Interaction Figure 2. Solution Structures of Tandem AT3-UIM12 in Free and Ub-Bound Forms. A, Ensemble of 10 structures superimposed on the polypeptide backbones of the a-helix of UIM1. B, Ensemble of 10 structures superimposed on those of UIM2. The helical portions of AT3-UIM12 show relative high definition, but the linker region is unstructured and flexible. No tertiary contacts between helices are observed. C, Backbone superposition of 10 lowest-energy structures of AT3-UIM12 in the Ub-bound form. D, Ribbon representation of AT3-UIM12 structure in the Ub-bound form. The structure of the Ub-bound form includes two a-helices, helix-1 (residues 226–238) and helix-2 (residues 247–256), and a linker loop region (residues 239–244) between them. The figures were generated using MOLMOL. doi:10.1371/journal.pone.0013202.g002 UIM motifs has little contribution to the overall structure definition. Due to the flexibility of the linker region, the average root-mean- square deviations (RMSDs) for the ten structures for backbone and all heavy atoms are relatively high, though the two helical regions have reasonable RMSD values (Supplemental Table S1). The relative independence of the helical orientation provides a large interface for each helical motif to bind with the globular Ub molecule individually; and one AT3-UIM12 can potentially bind with two Ub molecules. To understand the binding mode of AT3-UIM12, we elucidated the structure of AT3-UIM12 complexed with Ub in solution. Upon binding with Ub, the chemical shifts of AT3-UIM12 change significantly to a higher dispersion (Supplemental Fig. S1B). We assigned the chemical shifts (BioMagResBank database accession number 16405) and the intra-molecular NOEs, and solved the solution structure of AT3-UIM12 in complex with Ub (Supple- mental Table S1) (Protein Data Bank accession code 2KLZ). To our surprise, the Ub-bound AT3-UIM12 displays a compact structure with typical helix-loop-helix folding pattern (Fig. 2C and 2D). There are hydrophobic contacts between the two a-helices, for example, Leu233 in helix-1 with Ile253 in helix-2. Compared with the free form that has a flexible linker (RMSD of 1.36), the Ub-bound form becomes relatively rigid with a marginally acceptable RMSD of 0.72. Three residues (Ser236, Arg237 and Gln238) contribute to formation of the longer helix-1, whereas two residues (Glu245, Glu246) are not involved in helix-2. This suggests that Ub binding causes allosteric effects on the structure of AT3-UIM12. Backbone Dynamics of AT3-UIM12 Complexed with Ub The structural analysis suggests that the Ub binding induces conformational arrangement of AT3-UIM12 to a more rigid structure, especially in the linker region. To further ascertain the intrinsic Ub-binding properties, we compared the backbone dynamics of AT3-UIM12 in the presence and absence of Ub. The 15N-{1H}-NOEs of the backbone amides of AT3-UIM12 in the free and Ub-bound forms were measured (Supplemental Fig. S2), and then the relative NOE intensities for the backbone amides in both forms were delineated versus amino acid sequence of AT3- UIM12 (Fig. 3). In general, the backbone amides with large 15N-{1H}-NOE values (0.7–1.0) are expected to have lower flexibilities and to be located in the rigid region, while those with small NOE values (,0.6) are considered to be located at the loops or linker regions that have high flexibility [30]. Due to no defined tertiary structure in the free form of AT3-UIM12, the entire NOE values are relatively low as compared with those in well-folded proteins. The residues in the two helical regions (226–235, 245–256) have PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13202