E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to persulfidation (2022)
Figure 3

Figure 3. Urmylation is an E2/E3-independent, and primarily lysine-directed protein conjugation.

Comparison of the individual Ahp1-Urm1 units in the asymmetric unit. Close up of the formed peptide bond with all nearby residues labeled and highlighted in a balls-and-sticks representation (Cys60 in red). The last strand of the attached Urm1 protein is shown in cartoon representation and the refined 2Fo-Fc density (blue) and the Fo-Fc omit map lacking the highlighted residues (green) is shown around Ahp1Lys63 and Urm1Gly111 at 1σ and 3σ, respectively.

Top: MS/MS spectrum of the K63-ε-GGH containing peptide from CtAhp1C30S and K32-ε-GGH containing peptide from ScAhp1C31S. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ions are labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. m/z: mass to charge ratio. Bottom: Schematic summary of the detected conjugated peptides by mass spectrometry for CtAhp1C30S, CtAhp1C30S K63A and ScAhp1C31S. The thickness of the line represents the number of replicates where the respective site was detected, and the height represents the associated maximum Mascot ion score (n≥3). The urmylation was identified as HisGlyGly tag on lysines, serines and threonines while persulfidation was searched as a mass shift: +32 Da (persulfidation) or +89 Da (persulfidation + carbamidomethylation) on cysteines.

Analysis of conjugation between CtAhp1C30S variants in combination with thiocarboxylated CtUrm1C55S in the presence of TBH. Unconjugated Ahp1 and Urm1 as well as the formed conjugates (Ahp1-Urm1) are indicated on the right.

Analysis of conjugation between ScAhp1C31S variants in combination with thiocarboxylated ScUrm1 in the presence of TBH. Unconjugated Ahp1 and Urm1 as well as the formed conjugates (Ahp1-Urm1) are indicated on the right. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.

TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.

In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.

Comparison of the crystal structures of the oxidized dimeric forms of Ahp1 from CtAhp1C30S (left) and ScAhp1 (right; PDB ID 4DSR) (Lian et al, 2012). All lysine residues (green) are shown in the ball and sticks representation. Conjugated lysine residues detected by mass spectrometry (in B) are labelled and highlighted in black. Other lysine residues are labeled in grey and peroxidatic cysteines (Cys60 in CtAhp1 and Cys62 in ScAhp1) and their surroundings are labeled in red.