TY - JOUR
T1 - A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome
AU - Huber, Eva M.
AU - Heinemeyer, Wolfgang
AU - Li, Xia
AU - Arendt, Cassandra S.
AU - Hochstrasser, Mark
AU - Groll, Michael
N1 - Funding Information:
The staff of the beamline X06SA at the Paul Scherrer Institute, Swiss Light Source, Villigen, Switzerland is acknowledged for assistance during data collection. The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007–2013) under BioStruct-X (grant agreement No. 283570). We are most grateful to Richard Feicht for the professional purification and crystallization of yeast proteasomes. This work was supported by the SFB 1035/A2 to M.G. as well as the U.S. National Institutes of Health grants GM046904 and GM083050 to M.H.
PY - 2016/3/11
Y1 - 2016/3/11
N2 - Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.
AB - Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.
UR - http://www.scopus.com/inward/record.url?scp=84960840621&partnerID=8YFLogxK
U2 - 10.1038/ncomms10900
DO - 10.1038/ncomms10900
M3 - Article
C2 - 26964885
AN - SCOPUS:84960840621
SN - 2041-1723
VL - 7
JO - Nature Communications
JF - Nature Communications
M1 - 10900
ER -