Differential interaction of the E3 ligase parkin with the proteasomal subunit S5a and the endocytic protein Eps15.
Parkin is a multidomain E3 ligase associated with autosomal recessive Parkinson disease. The N-terminal ubiquitin-like domain (Ubld) of parkin functions with the S5a proteasomal subunit, positioning substrate proteins for degradation. In addition the parkin Ubld recruits the endocytotic protein Eps15, allowing the E3 ligase to ubiquinate Eps15 distal from its parkin-interacting site. The recognition sequences in the S5a subunit and Eps15 for the parkin Ubld are ubiquitin-interacting motifs (UIM). Each protein has two UIM sequences separated by a 50-residue spacer in S5a, but only approximately 5 residues in Eps15. In this work we used NMR spectroscopy to determine how the parkin Ubld recognizes the proteasomal subunit S5a compared with Eps15, a substrate for ubiquitination. We show that Eps15 contains two flexible alpha-helices each encompassing a UIM sequence. The alpha-helix surrounding UIM II is longer than that for UIM I, a situation that is reversed from S5a. Furthermore, we show the parkin Ubld preferentially binds to UIM I in the S5a subunit. This interaction is strongly diminished in a K48A substitution, found near the center of the S5a interacting surface on the parkin Ubld. In contrast to S5a, parkin recruits Eps15 using both its UIM sequences resulting in a larger interaction surface that includes residues from beta1 and beta2, not typically known to interact with UIM sequences. These results show that the parkin Ubld uses differential surfaces to recruit UIM regions from the S5a proteasomal subunit compared with Eps15 involved in cell signaling.
Safadi
Susan S
SS
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
Shaw
Gary S
GS
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D020125:Mutation, Missense; D020734:Parkinsonian Disorders / Q000201:enzymology / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015398:Signal Transduction / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C498506:PSMD4 protein, human; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex