N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex

Abstract

Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

Figure 1. Ubc12 is N-terminally acetylated in eukaryotic cells

A, LC-MS/MS spectrum from endogenous hUbc12’s N-terminal peptide after Glu-C digestion/desalting, indicating XCorr and ΔCN values, y (red) and b (blue) ions used to match peptide sequence. B, In vitro bacterially-expressed yNatC reactions with [¹⁴C]-Acetyl-CoA and Ubc12^Met (free N-terminus) or Ubc12^AcMet (pre-acetylated negative control). C, MaxEnt LC-TOF spectra of yUbc12-His₆ purified from WT orΔmak3 yeast, or from coexpression with yNatC in E. coli. D, Immunoblot of yCul1 (Cdc53p) from indicated yeast strains. E, Genetic interactions between NatC subunit mak10 and cdc34-2.

Figure 2. Dependence of Dcn1 co-E3 activity on Ubc12 N-acetylation

A, Pulse-chase [³²P]~yNedd8 transfer from yUbc12 variants to yCul1 C-terminal domain (yCul1^C) complexed with Rbx1 −/+ yDcn1^P. B, Same as A, but with human proteins. C, Thermodynamic parameters for Ubc12 or its peptides binding to Dcn1^P and E1 by ITC. NB = no binding.

Figure 3. Dcn1^P recognition of Ubc12’s N-acetyl-methionine

A, hCul1^WHB (not shown)-hDcn1^P-AcetylhUbc12^1–15 structure, with hDcn1^P surface colored by conservation among human and yeast orthologs and Acetyl–hUbc12^1–15 peptide in cyan. B, Close-up of hUbc12’s acetylated N-terminus (cyan) binding hDcn1^P (salmon) in cartoon (left) or hUbc12’s N-acetyl-Met1 and residues 2 and 4 as spheres in a mesh view of hDcn1^P (right). C, Close-up of hUbc12’s acetylated N-terminus (cyan) binding hDcn1^P surface colored by electrostatic potential. D, Same as B, but with yeast proteins. E, Thermodynamic parameters for Ubc12 peptide binding to Dcn1^P by ITC. 5:9S refers to helical staple. *reference from 2C. NB = no binding. F, Solvent-exposure of helical staple in hUbc12 peptide (cyan) bound to hDcn1^P (surface, colored by electrostatic potential).

Figure 4. Structure-based Dcn1 mutant compensation for lack of Ubc12 N-terminal acetylation

A, Pulse-chase [³²P]-yNedd8 transfer from yUbc12^Met (top) or yUbc12^AcMet (bottom) to yCul1^C-yRbx1 with structure-based yDcn1^P mutants (note different time-courses). B, Thermodynamic parameters for binding between yDcn1^P or the Tyr190Ala mutant to unacetylated and acetylated yUbc12. *reference from 2C. C, Immunoblots for yCul1 (Cdc53p, top) or HA-tag (bottom) from mid-log whole cell extracts from Δdcn1 or Δdcn1/Δmak10 yeast harboring empty, WT Dcn1-HA, or Y190A Dcn1-HA expression vectors.