Abstract
The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. By deamidating Gln40 of Nedd8 to glutamate (Q40E), the bacterial cycle-inhibiting factor (Cif) family is able to inhibit CRL E3 activities, thereby interfering with cellular functions. Despite extensive structural studies on CRLs, the molecular mechanism by which Nedd8 Gln40 deamidation affects CRL functions remains unclear. We apply a new quantitative cross-linking mass spectrometry approach to characterize three different types of full-length human Cul1–Rbx1 complexes and uncover major Nedd8-induced structural rearrangements of the CRL1 catalytic core. More importantly, we find that those changes are not induced by Nedd8(Q40E) conjugation, indicating that the subtle change of a single Nedd8 amino acid is sufficient to revert the structure of the CRL catalytic core back to its unmodified form. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs.
Highlights
The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein modification
The cycle-inhibiting factors (Cifs) found in many pathogenic Gram-negative bacteria can irreversibly deamidate a specific glutamine residue (Gln40) of Nedd[8] and convert it to glutamate[15]. This Q40E modification has no effect on cullin neddylation, but can effectively abolish the E3 activity of CRLs and affect proper cullin deneddylation by the COP9 signalosome[15,16,17,18]. These observations raise an intriguing question as to how the subtle change of a single Nedd[8] amino acid is able to negate the effect of neddylation in remodelling the B100-kDa CRL catalytic core
We have determined that d0- and d10DMDSSO reacted with Cul1–Rbx[1] complexes with similar efficiency as illustrated in Supplementary Fig. 1c, reflected in previous testing on standard proteins[31]. These results demonstrate that d0- and d10-labelled DMDSSO are well suited for quantitative XL-MS (QXL-MS) analysis of these protein complexes
Summary
The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. The RING-finger domain of Rbx[1] is released from the pocket, deemed the ‘open’ state, but remains tethered by its N-terminus to Cul[5], presumably allowing the extended RING-finger to sample the three-dimensional (3D) space around Cul[5] This conferred flexibility has been proposed to enable Rbx[1] to close the distance between substrate and E2, facilitating the transfer of ubiquitin from E2 to substrate protein. The cycle-inhibiting factors (Cifs) found in many pathogenic Gram-negative bacteria can irreversibly deamidate a specific glutamine residue (Gln40) of Nedd[8] and convert it to glutamate[15] This Q40E modification has no effect on cullin neddylation, but can effectively abolish the E3 activity of CRLs and affect proper cullin deneddylation by the COP9 signalosome[15,16,17,18]. In combination with quantitative analysis, XL-MS can determine dynamic conversion between the average states of protein complexes under different conditions
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