An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

TY - JOUR

T1 - An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

AU - Papaleo, Elena

AU - Ranzani, Valeria

AU - Tripodi, Farida

AU - Vitriolo, Alessandro

AU - Cirulli, Claudia

AU - Fantucci, Piercarlo

AU - Alberghina, Lilia

AU - Vanoni, Marco

AU - De Gioia, Luca

AU - Coccetti, Paola

PY - 2011/5

Y1 - 2011/5

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Anaphase-Promoting Complex-Cyclosome

KW - Humans

KW - Molecular Dynamics Simulation

KW - Molecular Sequence Annotation

KW - Molecular Sequence Data

KW - Phosphorylation

KW - Principal Component Analysis

KW - Protein Structure, Tertiary

KW - Recombinant Proteins

KW - Saccharomyces cerevisiae Proteins

KW - Thermodynamics

KW - Ubiquitin

KW - Ubiquitin-Conjugating Enzymes

KW - Ubiquitin-Protein Ligase Complexes

U2 - 10.1371/journal.pcbi.1002056

DO - 10.1371/journal.pcbi.1002056

M3 - Journal article

C2 - 21637798

SN - 1553-734X

VL - 7

SP - e1002056

JO - P L o S Computational Biology (Online)

JF - P L o S Computational Biology (Online)

IS - 5

ER -