PolyUbiquitin chain linkage topology selects the functions from the underlying binding landscape

Abstract

Ubiquitin (Ub) can generate versatile molecular signals and lead to different celluar fates. The functional poly-valence of Ub is believed to be resulted from its ability to form distinct polymerized chains with eight linkage types. To provide a full picture of ubiquitin code, we explore the binding landscape of two free Ub monomers and also the functional landscapes of of all eight linkage types by theoretical modeling. Remarkably, we found that most of the compact structures of covalently connected dimeric Ub chains (diUbs) pre-exist on the binding landscape. These compact functional states were subsequently validated by corresponding linkage models. This leads to the proposal that the folding architecture of Ub monomer has encoded all functional states into its binding landscape, which is further selected by different topologies of polymeric Ub chains. Moreover, our results revealed that covalent linkage leads to symmetry breaking of interfacial interactions. We further propose that topological constraint not only limits the conformational space for effective switching between functional states, but also selects the local interactions for realizing the corresponding biological function. Therefore, the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity. The simulation results also provide several predictions that qualitatively and quantitatively consistent with experiments. Importantly, the K48 linkage model successfully predicted intermediate states. The resulting multi-state energy landscape was further employed to reconcile the seemingly contradictory experimental data on the conformational equilibrium of K48-diUb. Our results further suggest that hydrophobic interactions are dominant in the functional landscapes of K6-, K11-, K33- and K48 diUbs, while electrostatic interactions play a more important role in the functional landscapes of K27, K29, K63 and linear linkages.

Figure 1. Experimental structures of diUbs with different linkages.

Only five linkage types have been structurally characterized (summarized in Table 1 in Text S1). The corresponding structures are shown with the distal Ub unit (contributes a carboxyl group of G76 to form the linkage) in yellow and the proximal Ub unit (contributes an -amino group of lysine) in red, above a schematic cartoon. The formation of Ub interfaces is mainly contributed by two hydrophobic patches. One is the I44 patch (color in blue) consisting of L8, I44, V70, another is the I36 patch (colored in green) involving L8, I36, L71 and L73. These experimental structures include: compact structure of K6-linked diUb (2XK5, 3ZLZ), compact structure of K11-linked diUb (3NOB, 2XEW), open and compact structures of M1-linked diUb (2W9N, 3AXC, respectively), open and compact structures of K63-linked diUb (2JF5 and 3H7P, 3DVG, respectively), and four distinct structures of K48-diUb consisting of open (1F9J), closed (1AAR) and two compact conformations (1TBE, 3AUL, 3NS8 and 2PE9, respectively).

Figure 2. Compact conformations of diUbs preexist on the binding landscape of free Ubs.

The free energy surfaces as a function of the distance between the center of mass of Ub monomers () and RMSDs from the compact structures (PDB 3AXC, 2XK5, 3NOB, 1AAR and 3DVG) of five linkage types resolved by X-ray crystallography and NMR (M1, K6, K11, K48 and K63, listed in Table 1 in Text S1). The native conformational regions are labelled by grey in the free Ub model. Note that the same conformational space sampled by the free Ub model at given concentrations (5 mM here) was used. For comparison, the results of corresponding linkage models (CGM1, CGK6, CGK11, CGK48 and CGK63 models, see Table 2 in Text S1) are also plotted below. The compact structures of these linkage types are shown above with the distal Ub unit in yellow and the proximal Ub unit in red. The two hydrophobic patches, I36 and I44, are colored in green and blue, respectively.

Figure 3. Symmetry of interfacial interactions present in the free model is broken in the linkage models.

It shows the distribution of average interfacial contacts along the residue index of proximal Ub (black) and distal Ub (grey). The distribution was mapped onto the surface of two Ub monomers whose positions are artificial for better view of the binding surface. The distal unit is represented by light grey surface with yellow cartoon, while the proximal unit by dark grey surface with red cartoon. The “hot spot” residues taking part in the binding are highlighted by surface with colors from blue to red, corresponding to having low and high interfacial contacts, respectively. Note that the analysis was based on the conformations with 3.2 nm.

Figure 4. Relationship between conformational populations and interfacial interactions (hydrophobic and electrostatic interactions).

The interfacial potential energy is decomposed into hydrophobic energy and electrostatic energy . The energy distribution of average and of all eight linkage types is shown. The population distribution of open, closed, compact states is represented by magenta, orange and black pies, respectively. The compact state (black) is further decomposed into the I36I36 (red), I36I44 (blue) and other states (green). See quantitative results in Table 1.

Figure 5. Multi-basin functional landscape of K48-diUb can be used to well reconcile the seemingly contradictory experimental measurements of the conformational equilibrium.

(A) Free energy profile as a function of and RMSD from the X-ray structure of the closed form of diUb (). (B) Free energy profile as a function of the distance between I36 hydrophobic patches () and . Beside the closed and open basins (labeled by O and C), there are three intermediate basins (labeled by 1, 2, 3) on the free energy surface of K48-diUb. (C) Free energy profile as a function of . Intermediate states 2 and 3 are indistinguishable in the one-dimensional free energy profile. Open and closed populations are , and , respectively. The remaining conformational space is mainly consisting of three intermediate states whose population is about 70%.

Figure 6. A significant distinct free energy landscape of K11-diUb from that of K48-diUb.

(A) Free energy profiles as a function of and RMSD from the X-ray structure of the closed form of K48-diUb (). (B) Free energy profiles as a function of and RMSD from the X-ray structure of the compact form of K11-diUb (). (C) Free energy profiles as a function of the distance between I36 hydrophobic patches () and the distance between I44 hydrophobic patches () (D) Distribution of average interfacial contacts along the residue index of proximal Ub (black) and distal Ub (grey). Note that the results of K48-diUb model and K11-diUb model correspond to left and right subfigures, respectively.

Figure 7. Functional landscapes of K6-, K27-, K29- and K33-diUbs.

Upper subfigures are the free energy profiles of K6, K27, K29 and K33 linked diUbs plotted as a function of and . Lower subfigures are the free energy profiles of K11, K63 and K48 linkages, shown as references. They represent compact, open and the multi-state functional landscapes, respectively.

Figure 8. Schematic picture that the functional landscapes of different diUb chains are selected from the same binding landscape of two Ub monomers by corresponding topological constraint.

The binding landscape (grey) is a description of binding between two free Ub monomers, while the functional landscapes are a description of binding between two Ub units with an additional topological constraint, e.g. a peptide bond formed between proximal Ub and distal Ub. Two typical different topological constraints are represented by red and blue, respectively. Our simulation results reveal that the binding landscape contains most of functional states of diUbs with different linkage types. And part of these functional states are further stabilized by the corresponding linkages. Such as, the functional landscape of K48-diUb has a deeper basin of the I44-I44 conformational state which is also present on the binding landscape. But other functional states, such as compact conformation of K11-diUb and K63-diUb, cannot be sampled on the functional landscape of K48-diUb. In other words, these conformational space is forbidden as a consequence of specific topological constraints (grey and dark regions). The topological constraint limits the conformational space and selects the local interactions for realizing the corresponding biological function. Therefore, the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity.