Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.
11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.
Li
J
J
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132-0001, USA.
Gao
X
X
Ortega
J
J
Nazif
T
T
Joss
L
L
Bogyo
M
M
Steven
A C
AC
Rechsteiner
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000954:Antigens, Surface / Q000378:metabolism; D001324:Autoantigens; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D051847:Lithostathine; D008239:Lysine; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D010750:Phosphoproteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000954:Antigens, Surface; D001324:Autoantigens; D002135:Calcium-Binding Proteins; C099558:Ki antigen; D051847:Lithostathine; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine