Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes.
Lys(63)-linked polyubiquitin (poly-Ub) chains appear to play a nondegradative signaling and/or recruitment role in a variety of key eukaryotic cellular processes, including NF-kappaB signal transduction and DNA repair. A protein heterodimer composed of a catalytically active ubiquitin-conjugating enzyme (Ubc13) and its homologue (Mms2 or Uev1a) forms a catalytic scaffold upon which a noncovalently associated acceptor Ub and thiolester-linked donor Ub are oriented such that Lys(63)-linked poly-Ub chain synthesis is facilitated. In this study, we have used (1)H-(15)N nuclear magnetic resonance spectroscopy, in combination with isothermal titration calorimetry, to determine the thermodynamics and kinetics of the interactions between various components of the Lys(63)-linked poly-Ub conjugation machinery. Mms2 and Uev1a interact in vitro with acceptor Ub to form 1/1 complexes with macroscopic dissociation constants of 98 +/- 15 and 213 +/- 14 microM, respectively, and appear to bind Ub in a similar fashion. Interestingly, the Mms2.Ubc13 heterodimer associates with acceptor Ub in a 1/1 complex and binds with a dissociation constant of 28 +/- 6 microM, significantly stronger than the binding of Mms2 alone. Furthermore, a dissociation constant of 49 +/- 7 nM was determined for the interaction between Mms2 and Ubc13 using isothermal titration calorimetry. In connection with previous structural studies for this system, the thermodynamics and kinetics of acceptor Ub binding to the Mms2.Ubc13 heterodimer described in detail in this study will allow for a more thorough rationalization of the mechanism of formation of Lys(63)-linked poly-Ub chains.
McKenna
Sean
S
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Hu
Jing
J
Moraes
Trevor
T
Xiao
Wei
W
Ellison
Michael J
MJ
Spyracopoulos
Leo
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes
D015534:Trans-Activators; D014157:Transcription Factors; D025801:Ubiquitin; C471624:UBE2V1 protein, human; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D008239:Lysine