AT3-UIM/Ub Interaction Figure 8. Schematic Representation of the Tandem UIM Motifs with Various Forms of Ub Chains. A, Non-cooperative binding mode. A representative for the mode is the tandem UIM motifs in Vps27p [16], which bind with Ub independently and non-cooperatively. B, Avid binding mode. The tandem UIM motifs in RAP80 bind with K63-linked diUb by forming a long a-helical structure [19]. The predicted linker becomes rather rigid and may be preferable to the configuration of K63-linked diUb. C, Cooperative binding mode. A short linker region (such as in AT3-UIM12) tends to be involved in the cooperative binding with Ub chains or ubiquitinated substrates. This cooperation is also likely to be occurred in poly- or multiUb. When the tandem UIM motifs bind with any form of Ub, a conformational arrangement and dynamics change occur in the linker region that may favor Ub binding. The dashed line indicates that either mono- or diUb is involved in the binding mode, and Ubi denotes different forms of Ub, standing for mono-, di-, poly- or multiUb. doi:10.1371/journal.pone.0013202.g008 NMR Spectroscopy 15N- or 15N/13C-labeled samples (,1 mM) were dissolved in a PBS buffer (20 mM phosphate, 100 mM NaCl, pH 6.5) containing 8% or 100% D2O and 0.02% (w/v) NaN3. A mixture of labeled AT3-UIM12 and unlabeled Ub with a molar ratio of 1:4 was used for recording the spectra of Ub-bound form of AT3-UIM12. All the NMR experiments were performed at 25uC on a Varian UNITY INOVA 600 MHz spectrometer equipped with three RF channels and a triple-resonance pulsed-field gradient probe. The backbone and side-chain 1H, 15N and 13C resonances were assigned based on the spectra of 3D HNHA, HNCO, HNCACB, CBCA(CO)NH, CC(CO)NH, and 3D 15N TOCSY-HSQC, 13C HCCH-TOCSY. NOE distance restraints for structure calculations were obtained from 3D 15N-edited NOESY and 13C-edited NOESY (aliphatic 13C regions). Intermolecular NOEs between AT3-UIM12 and Ub were obtained from 3D F1 13C/15N-filtered-F3 15N-edited NOESY experiments as described [53]. A steady-state heteronuclear 15N-{1H}-NOE experiment [30] was performed for 15N-labeled tandem AT3-UIM12 in the presence and absence of Ub protein. The spectra for measuring the heteronuclear NOEs were recorded with a 2-second relaxation delay, followed by a 3-second period of proton saturation. In the absence of proton saturation, the spectra were recorded by a relaxation delay of 5 s. The NOE data were processed by using NMRPipe software [54] and analyzed by Sparky (T.D. Goddard and D.G. Kneller, Sparky 3, University of California, San Francisco). The steady-state 15N-{1H}- NOE enhancements were calculated as the ratio of peak intensities in spectra recorded with or without proton saturation. Structure Calculation and Analysis The CNS program [55] with the ARIA module [56] was adopted to assign NOE peaks and to calculate structures. The protein structures were assessed by using PROCHECK [57] and were displayed by the MOLMOL program [58]. Hydrogen bond restraints (two per hydrogen bond) were generated by a combination of H/D exchange data, medium-range NOEs, and chemical shift index. Backbone dihedral angle restraints (w and y) were derived from the TALOS program [59]. For AT3-UIM12, the restraints used for structure calculation are summarized in Table S1. The calculation in combination with iterative NOE peak assignments was performed for 9 cycles, and a total of 200 structures were finally obtained. Ten structures with the lowest energies, which exhibit no NOE violation .0.3 A˚ and no dihedral violation .5 A˚, were selected and displayed. NMR Titration Studies 15N-Ub at a molar concentration of ,0.2 mM was titrated with increasing amount of UIM peptides or its mutants. A series of PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13202