PDBsum entry 2nvu

Protein turnover, ligase

PDB id: 2nvu

Contents

Protein chains

530 a.a. *

789 a.a. *

176 a.a. *

76 a.a. *

Ligands

ATP

Metals

_MG

_ZN

Waters ×45

* Residue conservation analysis

PDB id:

2nvu

Name:

Protein turnover, ligase

Title:

Structure of appbp1-uba3~nedd8-nedd8-mgatp-ubc12(c111a), a trapped ubiquitin-like protein activation complex

Structure:

Nedd8-activating enzyme e1 regulatory subunit. Chain: a. Synonym: amyloid protein-binding protein 1, amyloid beta precursor protein-binding protein 1, 59 kda, app-bp1, protooncogene protein 1, hpp1. Engineered: yes. Maltose binding protein/nedd8-activating enzyme e1 catalytic subunit chimera. Chain: b.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: appbp1. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: ube1c, uba3. Gene: ube2m, ubc12. Gene: nedd8.

Resolution:

2.80Å R-factor: 0.241 R-free: 0.274

Authors:

D.T.Huang,H.W.Hunt,M.Zhuang,M.D.Ohi,J.M.Holton,B.A.Schulman

Key ref:

D.T.Huang et al. (2007). Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity. Nature, 445, 394-398. PubMed id: 17220875 DOI: 10.1038/nature05490

Date:

13-Nov-06 Release date: 30-Jan-07

Protein chain

Q13564  (ULA1_HUMAN) -  NEDD8-activating enzyme E1 regulatory subunit from Homo sapiens

Seq: 534 a.a.

Struc: 530 a.a.

Protein chain

Q8TBC4  (UBA3_HUMAN) -  NEDD8-activating enzyme E1 catalytic subunit from Homo sapiens

Seq: 463 a.a.

Struc: 789 a.a.*

Protein chain

P61081  (UBC12_HUMAN) -  NEDD8-conjugating enzyme Ubc12 from Homo sapiens

Seq: 183 a.a.

Struc: 176 a.a.*

Protein chains

Q15843  (NEDD8_HUMAN) -  NEDD8 from Homo sapiens

Seq: 81 a.a.

Struc: 76 a.a.

* PDB and UniProt seqs differ at 33 residue positions (black crosses)

Enzyme reactions

• Enzyme class 2: Chain B: E.C.6.2.1.64 - E1 NEDD8-activating enzyme.
• Reaction: ATP + [NEDD8 protein] + [E1 NEDD8-activating enzyme]-L-cysteine = AMP + diphosphate + [E1 NEDD8-activating enzyme]-S-[NEDD8 protein]-yl-L- cysteine

ATP (Bound ligand (Het Group name = ATP) corresponds exactly) + [NEDD8 protein] + [E1 NEDD8-activating enzyme]-L-cysteine = AMP + diphosphate + [E1 NEDD8-activating enzyme]-S-[NEDD8 protein]-yl-L- cysteine

• Enzyme class 3: Chain C: E.C.2.3.2.34 - E2 NEDD8-conjugating enzyme.
• Reaction: [E1 NEDD8-activating enzyme]-S-[NEDD8 protein]-yl-L-cysteine + [E2 NEDD8- conjugating enzyme]-L-cysteine = [E1 NEDD8-activating enzyme]-L-cysteine + [E2 NEDD8-conjugating enzyme]-S-[NEDD8-protein]-yl-L-cysteine

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.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1038/nature05490

Nature 445:394-398 (2007)

PubMed id: 17220875

Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.

D.T.Huang, H.W.Hunt, M.Zhuang, M.D.Ohi, J.M.Holton, B.A.Schulman.

ABSTRACT

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

Selected figure(s)

Figure 2.
Figure 2: Two Ubc12 binding sites depend on NEDD8(T) being thioester-linked to APPBP1–UBA3. a, Close-up view of the cryptic Ubc12-binding surface near the nucleotide-binding site, with the adenylation domain portion of UBA3 in pink, NEDD8(A) in lime, and Ubc12 in cyan. b, Close-up view of direct interactions between Ubc12 (cyan) and NEDD8(T) (yellow).

Figure 4.
Figure 4: A thioester switch toggling E1–E2 interactions. E1 is blue/pink, corresponding to APPBP1/UBA3, with UBA3's UFD red. E2 is cyan. The first and second UBLs binding E1 are yellow and lime, respectively. Catalytic cysteines are green. a, UBL(T), thioester-bound to E1, clashes with E1's UFD in initial conformation. b, E1's UFD rotation unmasks cryptic E2-binding sites, allowing doubly UBL-loaded E1 to bind free E2. c, UBL transfer to E2's cysteine eliminates the UBL's covalent tether to E1. This removes E2-binding sites, and allows reversion to the alternative E1 conformation. d, Steric clashing between E1 and E2~UBL facilitates product release. Another activation cycle ensues.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2007, 445, 394-398) copyright 2007.

Figures were selected by an automated process.