Characterization of two polyubiquitin binding sites in the 26 S protease subunit 5a.
Ubiquitylated proteins are degraded by the 26 S protease, an enzyme complex that contains 30 or more unique subunits. One of these proteins, subunit 5a (S5a), has been shown to bind ubiquitin-lysozyme conjugates and free polyubiquitin chains. Using deletional analysis, we have identified in the carboxyl-terminal half of human S5a, two independent polyubiquitin binding sites whose sequences are highly conserved among higher eukaryotic S5a homologs. The sites are approximately 30-amino acids long and are separated by 50 intervening residues. When expressed as small fragments or when present in full-length S5a molecules, the sites differ at least 10-fold in their apparent affinity for polyubiquitin chains. Each binding site contains 5 hydrophobic residues that form an alternating pattern of large and small side chains, e.g. Leu-Ala-Leu-Ala-Leu, and this pattern is essential for binding ubiquitin chains. Based on the importance of the alternating hydrophobic residues in the binding sites and previous studies showing that a hydrophobic patch on the surface of ubiquitin is essential for proteolytic targeting, we propose a model for molecular recognition of polyubiquitin chains by S5a.
Young
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P
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Deveraux
Q
Q
Beal
R E
RE
Pickart
C M
CM
Rechsteiner
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D001704:Biopolymers / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000502:physiology; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000378:metabolism*
D001704:Biopolymers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D009113:Muramidase; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease