Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity.
Biological systems critically rely on selective and specific protein interactions, creating building blocks that cooperatively form the basis of functional complexity [1]. In particular, regulatory and signaling pathways, such as conjugation of ubiquitin (Ub) or similar ubiquitin-like modifiers (UBLs), depend on specific recognition of binding partners whilst discriminating against non-specific interactions [2], [3]. Ub becomes conjugated to substrates through the action of the E1-E2-E3 enzymatic system. This cascade follows a pyramidal hierarchy wherein two human Ub-activating enzymes (E1s) and over 30 human Ub-conjugating (E2) enzymes and hundreds of E3 Ub protein ligases cooperate to catalyze subsequent substrate modification [4], [5]. Whereas the Ub-E1 enzymes have different selectivity [6], [7], it is mainly the selective associations between E2 and E3 enzymes among thousands of possible interactions that are primarily responsible for effective Ub conjugation [2], [3], [8], [9]. The molecular basis for this selectivity is provided by surface residues of E2 enzymes and by “cross-braced”, zinc-binding domains of the Really Interesting New Gene (RING) sub-family of E3 ligases [2]. These RING finger domains generally mediate binding with E2s through their highly conserved UBC-fold, although exceptions have been described [8], [10]. This fold is a strictly conserved structure surrounding the active site cysteine that covalently accommodates the activated Ub [8], [11], [12]. Regarding the E2 enzymes, the major structural determinants for E3 binding have been identified in residues located in helix 1 (H1), loop 1 (L1) and loop 2 (L2) within the classical E2-E3 interaction interface [10]. Efficient ubiquitin-conjugation depends on specific interactions between E2 and E3 enzymes. The high degree of sequence and structure conservation in E2s and, to some extent, among the E3 RING-finger domains and their reported E2-E3 interfaces, remains compatible with a highly selective binding for their cognate E2 or E3 partners [9], [13]. Conserved sub-families of E2 enzymes, such as those belonging to the UbcH5 branch, have been shown to cooperate with similar sub-sets of E3 ligases [13]. At the other side, structurally more divergent E2s display interactions with more specialized E3 enzymes, such as for example the APC/C-UbcH10 pair [14]. Furthermore, several studies have indicated that the ability of E2-E3 pairs to mediate biochemical Ub- or UBL-conjugation is directly dependent on the ability of physical E2-E3 interactions [15], [16]. Based on this, physical E2-E3 interactions can be regarded as primary determinants for efficient conjugation reactions. We demonstrate here that a subtle and minute change – an aspartic to glutamic acid substitution (one methylene group difference) – is sufficient to completely change the selectivity profile of an E2 enzyme toward E3 RING-finger domains. Using molecular modeling and molecular dynamics simulations, a network of intra-molecular salt-bridges was identified that controls the balance between a binding-competent and a binding-incompetent state. Perturbation of network components located both in and outside the classical interaction surface resulted in a switch in the E3 interaction binding profile. These results suggest a new and delicate mechanism of how protein-protein interaction selectivity is achieved within the promiscuous ubiquitination system.
The hypothesis of a side-chain equilibrium controlling UbcH8 and UbcH6 E2 interactions with E3 RING-finger domains was tested experimentally by perturbing key residues involved in the intra-molecular salt-bridge network. First, the significance of K117 was assessed in UbcH8, since K117 was predicted to play a central role in alternatively contacting D113 and D145, thereby, preventing D113 of interacting with the RING domain. By substituting UbcH8 K117 with a histidine, the E3 interaction pattern of UbcH8 was reverted to that of UbcH6 (Figure 4A). Histidine was chosen in order to maintain the total charge of the system. Note that, since the intracellular pH of yeast has been found to be around 5. 5 in most growth phases, it can be expected that the solvent-exposed histidine side-chains are protonated [20]. The result of this substitution is that the positively charged K117 side-chain is replaced by the shorter imidazole side-chain of histidine. This conservative mutation, K117H, does no longer allow a favorable salt-bridge formation with D113 due to the more rigid histidine side-chain, leaving the latter free to interact with the RING domain. This indeed yielded an interaction pattern that resembles that of UbcH6. In contrast, UbcH8 K117R preserves the UbcH8 interaction profile because arginine, which resembles lysine in terms of length and charge, is still able to bridge D113, shielding it away from interacting with the E3 (Figure 4A). These experimentally validated observations confirm the central role of K117 of UbcH8 as a regulator of the orientation of D113 and D145 and thereby as a regulator of RING interactions. We next investigated the role of D145, the most remote (as seen from the E3 interaction site) component of the network. Interaction screening of an UbcH8 D145K mutant demonstrates that D145K interacts in a similar manner as UbcH8 WT (Figure 4A). The introduced charge repulsion between D145K and K113 reinforces the bridging between K117 and D113, locking the latter into a “closed” salt-bridged conformation preventing RING-contacts. In contrast, the introduction of D145E in UbcH8 alters the interaction profile of UbcH8 WT to that of UbcH6 WT. Here, the longer glutamine side-chain can more effectively contact K117, forming a stable salt-bridge, and consequently freeing D113 for forming contacts with RING. To ascertain that this indeed involves D113, the double mutant UbcH8 D113A/D145E was also characterized, showing the loss of the previously gained E3 interactions (Figure 4A). These results also indicate that both ASP and GLU at position 113 are binding competent, provided they are not stabilized in their closed conformation by intra-molecular salt-bridge formation, highlighting again the key role of this salt-bridge network. Taken together, these results point toward a delicate network of intra-molecular salt-bridges that dynamically control the positioning of a crucial E3-interacting residue (schematically represented in Figure 4B). Residues located in the classical E2-E3 interface, as well as those that are more distantly located dynamically control the positioning of K117 that acts as a gatekeeper for the negatively charged hot-spot residue capable of mediating E3 interactions. While pH-dependent [21] or phosphorylation [22] regulatory mechanisms have been described previously, the subtle equilibrium identified here does not involve any change in net charge, but originates from a minimal difference of a single methylene group along a side-chain.
Although the catalytic UBC-fold of E2 enzymes is characterized by high levels of sequence and structural similarities, it is adapted to selectively recognize RING-finger domains to allow transfer of Ub to substrates [2], [23]. By comparing sequence and structure of two highly similar E2 enzymes with their global RING-finger interaction patterns, in combination with molecular dynamics simulations, we were able to identify an intra-molecular network of salt-bridges that actively control RING interactions. Perturbation of components of this dynamic network in combination with Y2H screening of interactions subsequently confirmed its crucial role in controlling the interaction specificity. Residue K109/K117 was identified to play a crucial role within this network. It binds in an exclusive manner either E105/D113 or D137/D145. This lysine appears to be strictly conserved among E2 enzymes. In UbcH5B, for example, the corresponding lysine residue at this position is the central K63, which has been intimately linked to UbcH5B selectivity within the UbcH5B-CNOT4 RING-interaction [15], [24]. Interestingly, UbcH5B K63E failed to interact with the WT CNOT4 RING-finger domain, but this could be fully restored by incorporating the charge-swapping D48K/E49K substitutions in CNOT4 [15]. Although K63 interacts directly with D48/E49, this is not the case for K109/K117, indicating that even among conserved residues there are different ways of involvement in generating selectivity and E2-E3 pairing. Additionally, the most remote component of the identified salt-bridge network, D137/D145 is also conserved in the E2 superfamily, but it is not directly involved in RING interactions. The demonstration in this work that mutations at this position can switch selectivity emphasizes that E2-E3 interaction specificity can be a consequence of a subtle, dynamic interplay between interface and non-interface residues. In the majority of E2 enzymes, residues involved in establishing E3 interaction specificity are not concentrated in a single hotspot, but dispersed over the N-terminal helix one and two relatively flexible and divergent loop regions (L1 and L2) [2], [11]. Mutagenesis of these residues can abolish or modulate E2-E3 specificity [10], [11], [15]. Apart from sequence information, additional structural characteristics are affecting RING selectivity, like the length of H1, the flexibility of L1 and L2 and the triangular distances between these elements [12], [24], [25]. Comparing the sequences of UbcH6 and UbcH8 reveals additional amino acid differences at several key positions in these regions. One of them, D58/E66, lies within H1 and it is not orientated toward the E2-E3 interface. D58/E66 is in close proximity to T144/152 and S146/154, which are solvent exposed and directly interacting with RING domains. Therefore, D58/E66 might affect E3 interactions either indirectly through T144/152 and S146/154 or via a repositioning of helix H1. Indeed, the linker region that connects H1 with the rest of the E2 enzyme is found to be flexible [26] and D58/E66 might be involved in a hinge-like mechanism that might allow some conformational freedom of H1, which influences the distances between the three triangle points and thereby RING-interactions. Finally, the N-terminal extension of the tested E2 enzymes, not present in the crystal structures, might also affect their E3 binding preference. This could explain why reverse mutations in UbcH6 that mimic UbcH8 did not restore the E3 interaction profile of UbcH8. The presence of another intermolecular salt-bridge in the UbcH6-TOPORS complex generated by HADDOCK, involving the residues LYS43 (UbcH6) and GLU28 (TOPORS) (data not shown), could explain why UbcH6 E105D did not affect the selectivity profile of UbcH6. This salt-bridge cannot be formed with UbcH8, where LYS43 is replaced by ALA51 in the sequence. Comparing the sequences of UbcH6 and UbcH8 indeed reveals amino acid differences at several key positions in this N-terminal extension known to be primarily involved in the interaction with the E1 ubiquitin-activating enzyme, but also to play a role in E3 interaction selectivity. We should note that Y2H screening of physical protein-protein interactions between E2 enzymes and E3 RING-finger domains does not directly address biological functionality, e. g. the ability to transfer the activated Ub. However, we previously demonstrated that E2-RING E3 interactions found by LexA-B42 yeast two-hybrid assays are good predictors for enzymatic functionality [13] and therefore believe that the results presented here are also relevant in a functional context. It is unlikely that minor alterations in side-chain characteristics described in this work would affect protein stability in vivo (and our in silico results are supporting this). In addition, a recent systematic analysis of interaction dynamics across different technologies reported that high-throughput yeast two-hybrid is the only available technology for detecting transient interactions on a large scale [27], which support the recourse to this technique to unravel labile E2-E3 interactions. Despite their high percentage of identity, it has been reported that UbcH6 plays a major role in ubiquitin-conjugation while UbcH8 has only a minor role in ubiquitination but rather is the key conjugating enzyme of the ISGylation pathway [17]. This functional difference is in line with our observations [13] and supports the importance of an E3 selectivity mechanism that must differenciate not only among Ub-E3 ligases but also between Ub and UbL E3 ligases. Finally, this unreported dynamic intra-molecular salt-bridges network constitutes a new fundamental principle to understand the structural and evolutionary determinants of multispecific recognition. In conclusion, a dynamic equilibrium of conserved residues in two highly homologous E2 enzymes was identified, that mediate RING interactions. Amino acids located both within the classical interaction surface as well as residues that are remote from this surface are actively involved in modulating side-chain conformations and thus availability for binding of crucial residues. The subtle and dynamic nature of the identified regulatory switch suggests new ways how protein interactions can be controlled. Furthermore, the observation that minimal sequence differences between two highly similar proteins can control protein interaction networks serves as a cautionary tale and raises new challenges for bioinformatics analysis and modeling of protein interactions. Finally, these findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship.
High-copy yeast-two hybrid (Y2H) shuttle plasmids expressing human UbcH6 (UBE2E1) and UbcH8 (UBE2E2) and RING-finger domains of 250 human RING-type E3 ubiquitin protein ligases as fusions with the E. coli LexA binding domain (BD) or with the B42 acidic activator domain (AD), respectively are described in [13]. All amino acid substitutions were introduced using QuickChange II Site-Directed mutagenesis (Stratagene), according to the manufacturers' instructions. Appropriate mutagenesis was validated using DNA sequencing. Both wild-type and mutant UbcH6 and UbcH8 were used as starting structures. Wild-type UbcH6 and UbcH8 structures were taken from the Protein Data Bank (PDB) (PDB-ID 3bzh and 1y6l, respectively). Selective mutations were introduced with CNS (Crystallographic and NMR System) [28], keeping the Ca and the Cb atoms fixed to preserve the side-chain rotamer. Each system was simulated in duplicate for 200 ns using the GROMOS G53a6 force field [29] and GROMACS 4. 5. 5 package [30]. The starting structures were solvated in a dodecahedral box with a minimal 14 Å distance between solute and box, resulting in systems of about 11500 SPC water molecules, 34 Na+ and 36 Cl− ions (±1, depending on the system). Simulations were performed under periodic boundary conditions at 300 K and constant pressure (1 bar), using either Berendsen [31] or v-rescale [32] coupling algorithms for temperature control and Berendsen [31] coupling algorithm for pressure control (with coupling constants of 0. 1 and 1. 0 ps, respectively). Bond lengths were constrained with the Linear Constraint Solver (LINCS) algorithm [33] and the time step for the integration was 2 fs. Electrostatic interactions were calculated using either the reaction-field method [34] with a 14 Å cut-off distance or Particle Mesh Ewald (PME) [35], [36] with a 10 Å cut-off distance. Non-bonded interactions were updated every 10 fs (with a 10 Å cut-off distance for the short-range neighbor list). MD analysis was performed excluding the first 1 ns of the trajectories. All various simulation settings resulted in similar trajectories and only the results obtained with PME and v-rescale temperature control are discussed here. Structural models of UbcH6 and UbcH8 in complex with the RING-finger domain of TOPORS were generated by structural alignment of single components onto the bound structures of existing E2-E3 complexes (PDB: 1fbv, 1ur6,3eb6,2c2v and 2oxq). The resulting models were subjected to a short refinement in explicit water using HADDOCK [18], [19]. Electrostatic potential surfaces were determined using the Adaptive Poisson-Boltzmann Solver package (APBS) [37] within Pymol (version 1. 3) [38]. Manipulation of yeast cells and two-hybrid techniques are described in [13]. Briefly, WT and mutant LexA-E2 fusions were co-transformed together with the pSH18-34 LacZ reporter plasmid in EGY48α cells. To evaluate the effects of E2 mutants on human RING-type interactions, 250 sequence-verified B42-RING constructs were arrayed in EGY48a cells. Mating between α and A cells was performed on non-selective Yeast Peptone Dextrose (YPD) -medium (24 hours at 30°C). Diploids were selected for 48 hours on synthetic complete (SC) medium lacking the amino acids histidine, tryptophan and uracil (HWU− medium) by manual transfer of yeast spots using a 384-well replicator pinning tool (V & P Scientific, San Diego). Putative interactions were assessed in triplicate by growing diploids on SC HWU− medium supplemented with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) or on SC HWUL− (idem as HWU− but also lacking leucine), including either galactose or glucose as main carbon source. Quantification of interactions was done after 72 hours as reported earlier [13].
During their life, proteins undergo various modifications ranging from structural marking or signaling to degradation. One major biochemical process involves ubiquitin, a small and evolutionary conserved protein. This regulatory protein serves as a tag that, when attached to a protein substrate, alters its function, cellular sub-location or commits the labeled protein to destruction in the proteasome. The high specificity of the ubiquitination pathway is achieved through interactions between two large protein families, E2 and E3, that ensure the efficient covalent conjugation of ubiquitin. By comparing two “almost identical” E2 enzymes, we identified a single minute substitution that, operated by a dynamic network of salt-bridges, functions as a subtle switch that controls interaction selectivity toward E3 proteins. Using a combination of bioinformatics and modeling techniques, complemented by mutagenesis and experimental screening of E2-E3 interactions, we unraveled an equilibrium between an “open”, binding-competent and a “closed”, binding-incompetent state. Subtle modifications in this network are sufficient to switch the selectivity profile. These findings should serves as a cautionary tale and raises new challenges for bioinformatics analysis, modeling and experimental engineering of protein-protein interactions. The dynamic nature of the identified regulatory switch suggests that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function.
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Dynamic Control of Selectivity in the Ubiquitination Pathway Revealed by an ASP to GLU Substitution in an Intra-Molecular Salt-Bridge Network