HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

In contrast, homology modeling and structural superpositions do not predict a direct role for residue D133 or residues QIT31-33 of ISG15 in the activation step (Figure 4 and 6a) . The D133 residue is part of a ridge of negatively charged residues along the ISG15 molecule [35] . The function of this ridge is unclear. D133 in HuISG15 corresponds to a negatively charged residue in Ub, SUMO, Nedd8, RUB1, Apg12 (all Asp residues) and Urm1 (occupied by a Glu residue). Models based on the AppBp1-Uba3-Nedd8-ATP complex or the Sae1-Sae2-SUMO-Mg-ATP complex, do not predict direct contact of residue 133 in ISG15 with Ube1L. The same hold true for residues 31-33. This suggests that mutations Figure 6 . Residue 89 in ISG15 is situated in the predicted contact area with its Activating enzyme. (A) ISG15 (red ribbons) overlaid to Nedd8 (green ribbons) bound to its Activating enzyme AppBp1-Uba3 (grey ribbons). Residue 89 (dark blue) of ISG15, makes contact with residues in the two b-strands (shown in brown) of the Activating enzyme that precedes its C-terminal domain. K193 in Uba3 (orange) is located very close to N89, and corresponds to W568 in human UbE1L. The other residues of ISG15 with an important effect on total ISGylation, situated at positions 133 (yellow) and 31-33 (cyan blue), do not make contact with UbE1L and are likely to be involved in another feature of the ISGylation process. (B) HuUbE1L was aligned with HuUbE1 and RhmUbE1L (Ensembl peptide sequence EN-SMMUP00000027609). The region 563-569 in HuUbE1L shows two substitutions (S565L and W568R, red coloured) in the corresponding region of RhmUbE1L, which better resembles the corresponding region in HuUbE1 (K607). doi:10.1371/journal.pone.0002427.g006 at position 31-33 and 133 influence total ISGylation by a different mechanism than mutation at position 89. This must occur later in the ISGylation process, since no effect of these mutations can be observed in the absence of the N89D mutation. Of note, notwithstanding their position on different Ub domains in ISG15, residues 31, 33 and 133 are situated at the same face of the molecule (see Figure 4 ) close to the electronegative ridge. As the total protein ISGylation results from the balance between conjugation and deconjugation, mutation of QIT31-33 to KIA and D133 to N in HuISG15 N89D conceivably could affect recognition or activity by (a) discriminate DUB(s), retaining the targeted protein in the ISGylated state. In line with this hypothesis, a role has been described for the N-terminal Ub fold of ISG15 in the efficiency of DUB recognition/activity of the SARS coronavirus papain-like protease [52] .