The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
Binding motif for UBA3 adenylation domain
Functional site description:
NAE1-UBA3 is the only known E1 to be involved in the NEDD8 cascade, which regulates cullin neddylation. Cullins are part of multi-subunit cullin-based E3s (CRLs), playing an important role in substrate ubiquitination and consequently regulated protein degradation. CRLs are activated by covalent attachment of NEDD8 to a conserved C-terminal lysine. First NAE1-UBA3 is recruited to one of the two NEDD8 E2s, UBE2M and UBE2F, to promote thiolester formation between E2 and NEDD8. The E2~NEDD8 intermediate is then bound by a DCNL via its PONY domain which mediates the recruitment of a E3 cullin subunit through binding to its WHB subdomain. The CRL RING domain (RBX1 or RBX2) acts as ligase by binding the E2~NEDD8 intermediate and catalysing NEDD8 transfer. The NAE1-UBA3 is required to be able to recognise both E2s which is in partly mediated by a distinct binding of the E2s core domain to the E1 ubiquitin fold domain and partly by a E2 N-terminal binding motif which binds to a groove of UBA3 in the E1 complex.
ELM Description:
Motif found in the N-terminal regions of NEDD8 E2s UBE2M and UBE2F, which mediates binding to NAE1-UBA3 at its UBA3 adenylation domain. Additionally a distinct binding of the E2s core domain to the E1 ubiquitin fold domain (ufd) takes place, involving two main hydrophobic clusters, with UBE2F forming one ionic interaction and UBE2M forming numerous. Both the E2 N-terminal binding peptide and the E2 core domain must bind UBA3 simultaneously for ideal transfer of NEDD8 from E1 to E2. The interaction between NAE1-UBA3 and the E2 N-terminal peptides are probably unique to the NEDD8 pathway, because many E2s from other UBLs lack N-terminal extensions.
The UBA3 docking groove binds the N-terminal E2 sequences showing a common motif characterised by a ϕ-ϕ-x-ϕ pattern. All three hydrophobic residues are conserved among the NEDD8 E2s family members (Leu4, Phe5 and Leu7 on UBE2M, Met1, Leu2 and Leu4 on UBE2F) from different species, however mutations of the second and forth positions of the motif have the greatest impact on binding. Ser6 on UBE2M forms a hydrogen bond with Ser313 on UBA3. The equivalent residue on UBE2F (Thr3) probably also makes this interaction. A lysine residue, which is spaced 0-4 amino acids from the polar Ser or Thr, forms hydrogen bonds with the carbonyls from UBA3's Arg136 and Phe138. UBE2M's Met1 tucks into a hydrophobic cavity on UBA3. Thus UBE2M has a longer interacting peptide chain than UBE2F. Furthermore there is a minimum length requirement between NEDD8 E2s docking peptide and its core domain, to ensure optimum binding. Because the motif is overlapping with the DCNL binding motif on NEDD8 E2s, and is in fact identical in terms of residues and position in the case of UBE2F, this peptide segment must be part of a switching mechanism during neddylation.
Pattern: [ILM][ILMF].{1,2}[ILM].{0,4}K
Pattern Probability: 0.0011962
Present in taxon: Eukaryota
o See 2 Instances for LIG_UBA3_1
o Abstract
Ubiquitination is a post-translational modification, which leads to the degradation of a substrate protein at the 26S proteasome. The conjugation of a Ubiquitin to its substrate involves a three-step multi-enzymatic process using an E1-(activating), E2-(conjugating) and E3-(ligase) enzyme (Brown,2015). One of the two main classes of E3 ubiquitin ligases contains a RING domain (either RBX1 or RBX2) that associates with a cullin subunit, which is why they are termed cullin/RING ligases (CRLs) (Petroski,2005).
CRLs are activated by covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal lysine. This process is called neddylation and prevents the association of a CRL with its inhibitor CAND1 (cullin-associated NEDD8-dissociated 1).
Analogous to ubiquitination, neddylation also involves a three-step enzymatic process. The NEDD8 E1 is a heterodimer composed of NAE1 and UBA3. NAE1-UBA3 interacts with two known NEDD E2s, UBE2M and UBE2F. NEDD8 E3 ligases are represented by the RING domains of CRLs. Cullins bind substrate recognition subunits, the RING domains acts as ligases by binding the E2~NEDD8 intermediate to catalyse NEDD8 transfer. UBE2M preferentially interacts with the E3 RBX1 and UBE2F with RBX2 (Brown,2015). RBX1 can interact with CUL1-4, whereas RBX2 exclusively interacts with CUL5, leading to UBE2F specificity for CUL5 (Monda,2013).
Defective in cullin neddylation protein 1-like proteins 1-5 (DCNL1-5), are scaffold-like co-E3s that regulate cullin neddylation by acting as cofactors for RBX1 and RBX2.
All essential functions of DCNLs for neddylation reside within the PONY domain. DCNLs bind an E2~NEDD8 intermediate via their PONY domain and mediate the recruitment of an E3 cullin subunit through binding to its WHB subdomain (Kurz,2008). Also, the PONY domain contains the binding site for the UBE2M and UBE2F N-terminal motifs, based on a conserved ^M[IL].L core sequence. These peptides bind into a deep conserved hydrophobic pocket of DCNLs PONY domain (Monda,2013, 3BQ3, 3TDU, 4GAO, 4GBA). The binding motifs are overlapping, with the motifs responsible for NEDD8 E2~E1 interactions (LIG_UBA3_1), in fact in the case of UBE2F the motif residues are identical. A motif characterised by a conserved ϕ-ϕ-x-ϕ pattern can be found in UBE2M and UBE2f across species (ϕ, hydrophobic residue; x, any residue).
The NAE1-UBA3 is required to be able to recognise both E2s which is in partly mediated by a distinct binding of the E2s core domain to the E1 ubiquitin fold domain and partly by a E2 N-terminal binding motif which binds to a groove of UBA3 in the E1 complex and selects for the NEDD8 pathway. Multiple E1-E2 interaction surfaces may serve in increasing affinity, while remaining adaptable to both E2 but at the same time preventing any mischarges of the wrong E2 (9250909, 3FN1, 1TT5). It was proven, that both the E2 N-terminal binding peptide and the E2 core domain must bind UBA3 simultaneously for optimal transfer of NEDD8 from E1 to E2. The interaction between NAE1-UBA3 and the E2 N-terminal peptides are probably unique to the NEDD8 pathway, because many E2s from other UBLs lack N-terminal extensions. Because the N-terminal extention of UBE2M was shown to play an important role in mitogenisis in CSF-1 dependent proliferation, NAE1-UBA3 and the E2s may serve as good targets for agents with antimitotic effects (Huang,2004).
o 5 selected references:

o 4 GO-Terms:

o 2 Instances for LIG_UBA3_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q969M7 UBE2F
UBE2F_HUMAN
1 9 MLTLASKLKRDDGLKGSRTA TP 1 Homo sapiens (Human)
P61081 UBE2M
UBC12_HUMAN
4 12 MIKLFSLKQQKKEEESAGGT TP 4 Homo sapiens (Human)
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

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