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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., Author Summary A major mechanism for promoting protein regulation in eukaryotes involves the labeling with ubiquitin molecules of target proteins. Protein ubiquitination is involved in almost all aspects of eukaryotic cellular functions and is mediated, at the molecular level, by a hierarchical cascade of three different enzymes. Among these enzymes, E2 ubiquitin-conjugating enzymes are located at the heart of the ubiquitination pathway and are key mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Since several E2s have also been related to a variety of cancer and neurodegenerative disorders, increasing efforts are being devoted to the understanding of E2 regulation at the molecular level, a mandatory step for a complete understanding of the ubiquitination process. In the present contribution, we propose, by computational and biochemical investigations, a conserved mechanism of regulation by phosphorylation of the catalytic activity of a class of E2 enzymes, which plays a major role in the regulation of cell cycle progression and tumor development. Our results shed new light on and clarify molecular aspects related to one of the first steps of the ubiquitination cascade and its regulation.