Conformational dynamics and structural plasticity play critical roles in the ubiquitin recognition of a UIM domain

Abstract

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single alpha-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of (15)N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.

Figure 1. Solution structure of the Ubiquitin/UIM fusion protein

(a) An overlay of the 20 lowest energy conformations of the converged NMR ensemble (PDB ID 2kdi). A superposition of the x-ray structure of Ubiquitin (PDB ID iubq) is shown to illustrate perturbations in the structure induced by UIM binding. (b) Residue-specific structural statistics for the UIM/Ubiquitin complex. NOE constraint densities per residue (top) are compared to the ¹H-induced ¹⁵N NOE (middle) and residue-specific RMSD values for the NMR ensemble. Under restrained regions in the ensemble (bottom) coincide with the regions of increased plasticity (middle) as illustrated by reduced 1H-induced 15N NOE and NOE density. Residue numbering includes an initial 9 residue-long Histidine-containing sequence.

Figure 2. Structure diagram of UIM-Ubiquitin interactions

UIM binding is accommodated by a continuous hydrophobic patch on ubiquitin's solvent-exposed surface, formed by residues L8, V70, I44 and A56 shown in red from left to right in the structure. The UIM uses hydrophobic residues on two sides of the helical structure (shown as yellow and green sticks respectively) to engage the clamp-like surface on ubiquitin. Additional stabilizing electrostatic interactions are formed between the UIM n-terminal DEEE motif (red) and R42 and R72 on ubiquitin's β₃ strand and C-terminus (blue surface).

Figure 3. Backbone dynamics of ubiquitin decrease within the UIM interaction surface upon binding

Changes in backbone chemical shifts and fast timescale motions are shown. (a) sites that undergo chemical shift perturbations closely correlate with those that show a decrease in the generalized order parameter. Two opposite views of the molecule are shown, the top containing the UIM-binding interface. Colors are based on grouping according to upper and lower bounds of ppm distances and order parameter changes respectively, as indicated by the color bars in the corresponding graphs (b).

Figure 4. Summary of CPMG-derived exchange rates

As an indication of conformational exchange, the exchange contribution to the transverse relaxation rate, Rex, is shown for amide nitrogen atoms along the sequence of ubiquitin and fusion proteins (a). These values are derived as the difference between peak intensities obtained at zero and high (938Hz) CPMG frequencies. For comparison with the location the UIM binding interface, ¹⁵N chemical shift changes in the presence of the UIM are shown (b). No significant exchange contribution to R₂ was observed for most sites in ubiquitin (blue), while for the fusion protein the presence of conformational dynamics is manifested as high Rex values at several sites, for both the 600 and 800MHz data (green and red respectively). For Asp24, Rex values could not be accurately measured due to extended line broadening in both proteins, which indicates the presence of conformational exchange at the μsec-msec timescale.

Figure 5. Representative CPMG relaxation dispersion data

Effective transverse relaxation rates are shown as a function of the CPMG power, in units of Hz. For each site, independent fits of the modified form of the Carver-Richards equation (equation 1 in materials and methods) are also plotted. The fitted parameters for these sites can be found at table 2. Residues participating in similar conformational motions, in terms of the global parameters (k_ex and p_A,B) are presented in the same row. From these sites, Lys6, Ile44, Val70 and Leu96 are located on the interaction interface while Leu50 and Leu56 on the extended loop. For the ubiquitin without the UIM the corresponding curves are flat, indicating induced dynamics at the μsec to msec timescale upon UIM docking.

Figure 6. UIM-induced conformational dynamics in ubiquitin

The sites that participate in conformational exchange processes are shown on the structure of the fusion protein. Interactions with the UIM induce conformational motions on the docking interface residues in addition to a second coordination layer of atoms and distal sites located in the extended loop that connects strands 3 and 4. Sites are color-coded according to the exchange process they participate in. Red: Process a – reorganization of the interface; Blue: Process b – induced loop motions; Magenta – undecided. The sidechains of Lysines at positions 6 and 48 that are confirmed ubiquitylation sites are also shown. Raw relaxation data for a selection of these sites is shown in Figure 5 while fit parameters in table 2.