Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States.
Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).
Spyracopoulos
Leo
L
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. leo.spyracopoulos@ualberta.ca
Lewis
Michael J
MJ
Saltibus
Linda F
LF
D001665:Binding Sites; D003001:Cloning, Molecular; D014644:Genetic Variation; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes
D011994:Recombinant Proteins; D025801:Ubiquitin; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases