title: Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination.
abstract: Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(βTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Chong
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5CI;
authors.forename: Donald E
authors.initials: DE
authors.lastname: Spratt
authors.affiliation: Departments of Physiology and Biophysics, University of California, Irvine, CA 92697;
authors.forename: Yingying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Chan
authors.initials: C
authors.lastname: Lee
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Jaladhi
authors.initials: J
authors.lastname: Nayak
authors.affiliation: Department of Molecular Biology and.
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Rana
authors.initials: R
authors.lastname: Elkholi
authors.affiliation: Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and.
authors.forename: Inger
authors.initials: I
authors.lastname: Tappin
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Jessica
authors.initials: J
authors.lastname: Li
authors.affiliation: Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and j-hurwitz@ski.mskcc.org zhen-qiang.pan@mssm.edu.
authors.forename: Jerard
authors.initials: J
authors.lastname: Hurwitz
authors.affiliation: Genetics and Genomic Sciences, and.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Brown
authors.affiliation: Departments of Oncological Sciences.
authors.forename: Jerry Edward
authors.initials: JE
authors.lastname: Chipuk
authors.affiliation: Department of Molecular Biology andHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Zhijian J
authors.initials: ZJ
authors.lastname: Chen
authors.affiliation: Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029-6574;
authors.forename: Roberto
authors.initials: R
authors.lastname: Sanchez
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5CI;
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
authors.affiliation: Departments of Physiology and Biophysics, University of California, Irvine, CA 92697;
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
authors.affiliation: Departments of Oncological Sciences,Xuzhou Medical College, Jiangsu Key Laboratory of Biological Cancer Therapy, Jiangsu 221002, China j-hurwitz@ski.mskcc.org zhen-qiang.pan@mssm.edu.
authors.forename: Zhen-Qiang
authors.initials: ZQ
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000235:genetics; D055162:Biocatalysis; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044843:SKP Cullin F-Box Protein Ligases; D008239:Lysine
keywords: E2 ubiquitin-conjugating enzyme; E3 ubiquitin ligase; receptor ubiquitin