E2 enzymes define a complex superfamily that includes 17 families 
. They play a major role in protein ubiquitination and Ub chain assembly 
. Recently, E2s have been shown to be involved in a variety of disorders, including cancers and neurodegenerative diseases 
. Accordingly, there is increasing interest in understanding their regulation at the molecular level, a mandatory pre-requisite for rational design of effective pharmacologically active molecules which target E2 function.
An acidic insertion in the loop β2α4 has been identified in the proximity of the catalytic cysteine of several E2 enzymes, including Cdc34 that plays a major regulatory role in cell cycle progression and tumor development 
. The acidic loop has been thoroughly investigated by mutagenesis experiments. In fact, mutations of acid residues in the loop abolished the polyubiquitin chain assembly, the processivity and the synthesis of a polyubiquitin chain with a correct topology 
. It has been proposed that the loop is crucial for E2 downstream signaling such as interactions with E3 or correct interactions with the target substrates. Nevertheless, the 12-residue acidic insertion is not essential for yeast Cdc34 function in over-expression conditions 
and its role in Cdc34 function remains unexplained.
In this paper, we report that the loop acts as one element of a bipartite signature structure conserved among the Cdc34-like E2 enzymes belonging to family 3 and regulates enzyme activity through a phosphorylation mechanism. The second component of the signature structure is defined by serines within the catalytic domain, corresponding to S130 and S167 of yeast Cdc34, which are phosphorylated by CK2, a highly conserved protein kinase, essential in several different cellular processes. Together these two elements define a molecular switch that modulates opening and closing of the catalytic cleft, and whose dynamics can be finely regulated by CK2 phosphorylation (see below).
In the unphosphorylated protein, the β4α2 loop has high conformational freedom (, ). MD trajectories show that acidic residues of the loop came close to S130 of Cdc34 located in an α-helix forming one side of the catalytic cleft (-). These movements make the loop act as a “lid”, switching from “open” to “closed” conformations with respect to the catalytic cleft (). The mobility of the acidic loop in Cdc34UBC
and its modulation of solvent accessibility of the E2 catalytic cleft could explain the low basal Ub charging activity by unphosphorylated Cdc34 observed in vitro 
, also in agreement with the fact that the NMR structure of the human homolog Ube2g2, an E2 enzyme belonging to family 3, is characterized by conformations of the acidic insertion which in general does not provide accessibility of the catalytic cysteine 
Model of the activation mechanism of ubiquitin charging activity of Cdc34-like E2 enzymes.
Repulsive electrostatic effects between phospho-S130 and the acidic residues in the loop decrease its mobility, triggering an outward displacement of the loop and a competent conformation for Ub-charging (). The proposed mechanism for Cdc34-like E2s activation is also in agreement with preliminary homology models of complexes between Cdc34-like enzymes and Ub, in which only open conformations of the loop are compatible with Ub interactions in the catalytic cleft (data not shown
). It is in agreement as well with the previous observation that the acidic loop in a more closed conformation would result in steric clashes with the C-terminus of the ubiquitin molecule 
. Our proposed model further predicts that if the electrostatic repulsive effects, which promote conformational changes of the loop, are abolished by mutations of the phospho-sites or of the acidic residues in the insertion, the loop cannot be stabilized in an open conformation upon phosphorylation, compromising downstream events in the Ub pathway. Consistently with this notion, while Cdc34 Ub-charging activity is modulated by CK2-dependent phosphorylation of S130 (and S167, see later) 
, ubiquitin charging activity of His6
-Cdc34-Δ12 is unaffected by phosphorylation ().
It has also to be considered, in our model, that ubiquitin-charging of E2 enzymes requires an interaction between E2 and E1 enzyme and a transfer of ubiquitin from the E1 catalytic cysteine to the E2 catalytic cysteine. In order, to strengthen our model, we derived by similarity with experimentally known E1-E2 complexes, a model of the putative complex between Uba1 E1 enzyme and Cdc34 with both open and closed conformations of the acidic loop ( in Text S1
). This qualitative model suggests that the acidic loop can be accommodated in the E1 binding cavity both in the open and closed conformation without causing steric effects, but if the loop is in a closed conformation it is likely to create a barrier between the E1 and E2 catalytic cysteines and probably prevents the transfer of Ub molecule. As it can be judged from the Cdc34-E1 model, the presence at position 130 of Cdc34 of serine or phospho-serine does not significantly affect the intermolecular interaction network at E1-E2 interface, even if further calculations will be necessary to clearly define the intermolecular interactions in details, whereas Uba1-Cdc34 model emphasizes the notion that the prominent role of S130 phosphorylation is to promote the acidic loop displacement (data not shown
). Previous results showed that in vivo
both S130 and S167 residues need to be mutated to alanine to make the yeast Cdc34 protein unable to complement a cdc34-2ts
mutant, although evident morphological defects were observed in strains expressing Cdc34S130A
(but not Cdc34S167A
. Phosphorylation of S130 residue is likely to account for the most relevant conformational changes induced by the post-translational modification, whereas S167 may have additive enhancing effects as suggested by double-phosphorylated Cdc34 MD simulations. In our model, S167 is located close to the C-terminal end. As a result, constraints on S167, as well as its interactions with other protein domains are likely to be lost. A detailed computational study of the role of S167 phosphorylation will require a suitable structure of the full-length Cdc34 protein.
In a broader context, it has been established that post-translational phosphorylation is a widespread mechanism for the regulation of protein biological activity 
. In several cases, enzyme activity or protein function has been reported to be regulated by inhibitory or activatory phosphorylation events at specific protein sites. At the molecular level, these events are mediated by electrostatic repulsion between the phospho-residues and the neighboring negatively charged aminoacids 
, pointing out a general and important regulatory mechanism. These mechanisms can be successfully investigated in atomic details by MD simulations 
In conclusion, our study sheds a new and unexpected light on the role of the acidic loop in Cdc34-like E2 enzymes and provides the first evidence that this loop is crucial not only for downstream events related to Ub chain assembly 
, but above all for modulation of an upstream crucial step of the Ub pathway: the covalent Ub-binding of Cdc34-like E2s. The loop activation by phosphorylation of serine residues in the UBC domain is a mandatory step for an efficient Ub-charging and could also account for the proper proceeding of downstream events in the ubiquitin pathway.