title: Differential ubiquitin binding by the acidic loops of Ube2g1 and Ube2r1 enzymes distinguishes their Lys-48-ubiquitylation activities.
abstract: The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184-196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1(1-183) (Ube2r1C). Replacement of Gln-105-Ser-106-Gly-107 in the acidic loop of Ube2r1C (Ube2r1C(YGY)) by the corresponding residues from Ube2g1 (Tyr-102-Gly-103-Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C(C93S)-[(15)N]UB(K48R) oxyester displayed two-state conformational exchange, whereas the Ube2r1C(C93S/YGY)-[(15)N]UB(K48R) oxyester showed predominantly one state. Together with NMR studies that compared UB(K48R) oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, Department of Bio-Analytical Science, University of Science and Technology, Daejon 305-333, South Korea, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870.
authors.forename: Yun-Seok
authors.initials: YS
authors.lastname: Choi
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883.
authors.forename: Yun-Ju
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883.
authors.forename: Seo-Yeon
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington 98195, and.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, Department of Bio-Analytical Science, University of Science and Technology, Daejon 305-333, South Korea.
authors.forename: Jung-Hye
authors.initials: JH
authors.lastname: Ha
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883.
authors.forename: Hae-Kap
authors.initials: HK
authors.lastname: Cheong
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, Department of Bio-Analytical Science, University of Science and Technology, Daejon 305-333, South Korea.
authors.forename: Chaejoon
authors.initials: C
authors.lastname: Cheong
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Cohen
authors.affiliation: From the Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, Department of Bio-Analytical Science, University of Science and Technology, Daejon 305-333, South Korea, ksryu@kbsi.re.kr.
authors.forename: Kyoung-Seok
authors.initials: KS
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D025821:Polyubiquitin / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry; D054875:Ubiquitination
chemical_list: D004220:Disulfides; D004952:Esters; D025801:Ubiquitin; D025821:Polyubiquitin; C577447:CDC34 protein, human; C097309:UBE2D2 protein, human; C471615:UBE2G1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
keywords: Nuclear Magnetic Resonance (NMR); Structural Biology; Ubiquitin; Ubiquitin-conjugating Enzyme (E2 enzyme); Ubiquitylation (ubiquitination)