A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys63 ubiquitin conjugation complex.
Human Ubc13 and Mms2 (or its homolog, Uev1) form a unique ubiquitin-conjugating enzyme (Ubc) complex that generates atypical Lys(63)-linked ubiquitin conjugates. Such conjugates are attached to specific targets that modulate the activity of various cellular processes including DNA repair, mitotic progression, and nuclear factor-kappaB signaling. Whereas Ubc13 is a typical Ubc, Mms2 is a non-catalytic Ubc variant. Substantial biochemical evidence has revealed a mechanism whereby Mms2 properly orients ubiquitin to allow for Lys(63) conjugation by Ubc13; however, how this specific Ubc13-Mms2 complex is formed and why Mms2 does not form a complex with other Ubcs have not been reported. In order to address these questions, we used a structure-based approach to design mutations and characterize the human Ubc13-Mms2 interface. We used the yeast two-hybrid assay, glutathione S-transferase pull-downs, and surface plasmon resonance to test in vivo and in vitro binding. These experiments were paired with functional complementation and ubiquitin conjugation studies to provide in vivo and in vitro functional data. The results in this study allowed us to identify important residues of the Ubc13-Mms2 interface, determine a correlation between heterodimer formation and function, and conclude why Mms2 forms a specific complex with Ubc13 but not other Ubc proteins.
Pastushok
Landon
L
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Moraes
Trevor F
TF
Ellison
Michael J
MJ
Xiao
Wei
W
D000595:Amino Acid Sequence; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D025801:Ubiquitin; C471607:UBE2N protein, human; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D008239:Lysine