Abstract
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.
Highlights
Biological systems critically rely on selective and specific protein interactions, creating building blocks that cooperatively form the basis of functional complexity [1]
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]
Proteins undergo various modifications ranging from structural marking or signaling to degradation
Summary
Biological systems critically rely on selective and specific protein interactions, creating building blocks that cooperatively form the basis of functional complexity [1]. 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]. 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, exceptions have been described [8,10]. 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]
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