An Acidic Loop and Cognate Phosphorylation Sites Define a Molecular Switch That Modulates Ubiquitin Charging Activity in Cdc34-Like Enzymes

Abstract

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis

“…It is well known that amino acidic phosphorylation involves changes in protein stability and regulates affinity with other components. After protein phosphorylation, acidic loops formation has been described for other proteins (Papaleo et al, 2011). Cholesterol release could result from a pH-dependent transition to a molten globule structure, involving the loss of association between the C-terminal α-helix and lipid molecules in the OMM.…”

C-terminal acidic domain of ubiquitin-conjugating enzymes: A multi-functional conserved intrinsically disordered domain in family 3 of E2 enzymes

“…8), in light of our MD results and the available data in the literature (Kleiger et al, 2009;Papaleo et al, 2011;Sadowski et al, 2007;Spratt and Shaw, 2011) even if it has to be kept in mind that the MD ensemble here described cannot provide an exhaustive representation of the AD conformational ensemble, as aforementioned. In the free state, the AD domain can be characterized by a-helical structures and it can populate globular conformations, in which the residues likely to trigger the interactions with the cognate E3 (as the aforementioned tyrosine residues) or the Ub molecule (negatively charged residues in the N-terminal region of the domain, as E189, D200, E201 and the phospho-site S203) can be shielded from the solvent by transient tertiary contacts.…”

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

“…Ube2R1/2 was chosen for the following reasons: (i) it has critical importance for cellular homeostasis; (ii) it forms polyubiquitin chains with exquisite Lys 48 specificity (27); and (ii) previous modeling efforts elucidated the conformation of the Ube2R1-donor ubiquitin interface, which was subsequently confirmed by X-ray crystallography (30,34). The orientation of the acidic loop has been previously characterized by molecular dynamics (24) and was also included during the modeling procedure.…”

“…Ube2R1/2 is a critical E2 that functions with the cullin-RING ubiquitin ligases (1,18), and, together, these enzymes may be responsible for 20% of all proteasome-dependent degradation in human cells (19). Ube2R1/2 contains an atypical insertion distal to its active site that contains several conserved acidic residues (20)(21)(22)(23)(24)(25), and this acidic loop has also been shown to have an important role in Lys 48 selectivity (26,27). More recent work has identified a loop on acceptor ubiquitin that contains residues that interact with the E2 in order to help place Lys 48 in the E2 active site (26).…”