title: 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.
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy. elena.papaleo@unimib.it
authors.forename: Elena
authors.initials: E
authors.lastname: Papaleo
authors.forename: Valeria
authors.initials: V
authors.lastname: Ranzani
authors.forename: Farida
authors.initials: F
authors.lastname: Tripodi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitriolo
authors.forename: Claudia
authors.initials: C
authors.lastname: Cirulli
authors.forename: Piercarlo
authors.initials: P
authors.lastname: Fantucci
authors.forename: Lilia
authors.initials: L
authors.lastname: Alberghina
authors.forename: Marco
authors.initials: M
authors.lastname: Vanoni
authors.forename: Luca
authors.initials: L
authors.lastname: De Gioia
authors.forename: Paola
authors.initials: P
authors.lastname: Coccetti
mesh_terms: D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D006801:Humans; D056004:Molecular Dynamics Simulation; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D025341:Principal Component Analysis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; C077072:UBC7 protein, S cerevisiae; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome