PDBsum entry 5ifr

Transferase

PDB id: 5ifr

Contents

Protein chains

151 a.a.

76 a.a.

Ligands

GOL

Waters ×77

PDB id:

5ifr

Name:

Transferase

Title:

Structure of the stable ube2d3-ubdha conjugate

Structure:

Ubiquitin-conjugating enzyme e2 d3. Chain: a. Synonym: (e3-independent) e2 ubiquitin-conjugating enzyme d3,e2 ubiquitin-conjugating enzyme d3,ubiquitin carrier protein d3, ubiquitin-conjugating enzyme e2(17)kb 3,ubiquitin-conjugating enzyme e2-17 kda 3,ubiquitin-protein ligase d3. Engineered: yes. Polyubiquitin-b. Chain: b.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: ube2d3, ubc5c, ubch5c. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: ubb. Expression_system_taxid: 562

Resolution:

2.20Å R-factor: 0.194 R-free: 0.244

Authors:

J.N.Pruneda,M.P.C.Mulder,K.Witting,H.Ovaa,D.Komander

Key ref:

M.P.Mulder et al. (2016). A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nat Chem Biol, 12, 523-530. PubMed id: 27182664 DOI: 10.1038/nchembio.2084

Date:

26-Feb-16 Release date: 11-May-16

Protein chain

P61077  (UB2D3_HUMAN) -  Ubiquitin-conjugating enzyme E2 D3 from Homo sapiens

Seq: 147 a.a.

Struc: 151 a.a.*

Protein chain

P0CG47  (UBB_HUMAN) -  Polyubiquitin-B from Homo sapiens

Seq: 229 a.a.

Struc: 76 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class 2: Chain A: E.C.2.3.2.23 - E2 ubiquitin-conjugating enzyme.
• Reaction: S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine = [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L- cysteine
• Enzyme class 3: Chain A: E.C.2.3.2.24 - (E3-independent) E2 ubiquitin-conjugating enzyme.
• Reaction: S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E1 ubiquitin-activating enzyme]-L-cysteine + N₆- monoubiquitinyl-[acceptor protein]-L-lysine
• Enzyme class 4: Chain B: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1038/nchembio.2084

Nat Chem Biol 12:523-530 (2016)

PubMed id: 27182664

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

M.P.Mulder, K.Witting, I.Berlin, J.N.Pruneda, K.P.Wu, J.G.Chang, R.Merkx, J.Bialas, M.Groettrup, A.C.Vertegaal, B.A.Schulman, D.Komander, J.Neefjes, F.El Oualid, H.Ovaa.

ABSTRACT

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.