Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18

Abstract

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

Two NEMO-like Ubiquitin-Binding Domains in CEP55 Differently Regulate Cytokinesis

“…UBZ WRNP1 and UBZ RAD18 were chosen because their crystal structures showed a binding mode similar to that of ubiquitin, which is likely close to that of UBZ CEP55 . In addition, UBZ WRNP1 exhibits no ubiquitin linkage specificity, whereas UBZ RAD18 exhibits a little preference for K63 over K48 polyubiquitin (4-fold) and an in vitro stronger polyubiquitin binding, owing to the extended LR motif (224–240) located at its C terminus (Thach et al., 2015). We also used the UBZ NDP52 , which is also built on a ββα fold structurally related to UBZ WRNP1 and UBZ RAD18 .…”

“…A possible explanation could be the binding mode of UBZ RAD18 and its ubiquitin-linkage preference for K63 and linear chains over K48 chains, which appeared to be more similar to those of UBZ CEP55 . However, one cannot rule out the possibility that this best compensation effect was also due to the additional ELRM motif located at the C terminus of UBZ RAD18 (Thach et al., 2015), which can confer a better binding affinity to polyubiquitin chains compared with all the other UBZs and nZFs tested.…”

A novel mode of ubiquitin recognition by the ubiquitin‐binding zinc finger domain of WRNIP1

“…Intriguingly, an additional hydrogen bond is formed between Ala45 of WRNIP1 UBZ and Lys48 of ubiquitin. Unlike the C‐terminal residues, including Ala45 of WRNIP1 UBZ which makes a turn structure, the corresponding residues at the C‐terminus of Rad18 UBZ (residues 227–237) have been recently suggested to form an additional α‐helix and to interact with the proximal ubiquitin of the linear diubiquitin chain . These structural differences may explain the reason why Rad18 UBZ with C‐terminal extra residues shows linkage‐specific polyubiquitin binding while WRNIP1 UBZ does not show linkage specificity .…”

Genome-wide CRISPR screen identified Rad18 as a determinant of doxorubicin sensitivity in osteosarcoma

“…On the basis of previous studies, we speculate that there are two possible different mechanisms. On the one hand, Rad18 can associate with K63-linked Polyubiquitylated Chromatin proteins through the UBZ domain [ 49 , 56 ]. Therefore, Rad18 may promote the recruitment and assembly of MRN complexes at fragile DNA through a k63-ubiquitination dependent way.…”