Year-end Sale % OFF 
Abstract
Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.
Highlights
Ubiquitination controls a vast array of cellular functions in eukaryotes, including protein degradation, DNA repair, transcription, protein trafficking, the cell cycle and vesicle budding [1,2,3]
The connection between the ability of an E2 to form polyubiquitin chains and the presence of a non-covalent ubiquitin-binding site on the socalled ‘backside’ of the E2 was first discovered for UBCH5C [17,18], which interacts non-covalently with ubiquitin in a region centered on UBCH5C residue S22
Structural studies of the canonical backside of human UBCH5B [22], UBCH5C [17,47] and human RAD6B [18] have shown a common non-covalent ubiquitin binding site located on a similar region of the E2 (Figure 1D and E) and that is contacted by hydrophobic residue, I44, of ubiquitin [17,18,22,47]
Summary
Ubiquitination controls a vast array of cellular functions in eukaryotes, including protein degradation, DNA repair, transcription, protein trafficking, the cell cycle and vesicle budding [1,2,3]. Ubiquitin (Ub) is covalently attached to substrate lysine residues through the E1, E2 and E3 enzyme cascade [4,5,6]. Ubiquitin is transferred from the E1 to the active site cysteine of an E2 Ub-conjugating enzyme to form an E2∼Ub thioester [5]. In the case of RING E3 ligases, the RING domain binds to both the E2∼Ub thioester and the substrate, stimulating conjugation of the ubiquitin C-terminus to the ⑀-amino group of the target lysine [6]. Structural studies have provided snapshots of the different interactions that mediate each step in the ubiquitination cascade [4,5,6] our understanding of how differences among E2 enzymes govern substrate specificity remains incomplete
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
