1. Howard Hughes Medical Institute,
2. Departments of Structural Biology and Genetics/Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA
3. Interdisciplinary Program, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA
4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
5. Physical Biosciences Division, Lawrence Berkeley National Laboratory University of California, Berkeley, California 94720, USA

Correspondence to: Brenda A. Schulman^1,2,3 Correspondence and requests for materials should be addressed to B.A.S. (Email: brenda.schulman@stjude.org).

Abstract

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1–E2–E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1~UBL thioester intermediate, and generating a thioester-linked E2~UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1–UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1–E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

1. Howard Hughes Medical Institute,
2. Departments of Structural Biology and Genetics/Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA
3. Interdisciplinary Program, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA
4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
5. Physical Biosciences Division, Lawrence Berkeley National Laboratory University of California, Berkeley, California 94720, USA

Correspondence to: Brenda A. Schulman^1,2,3 Correspondence and requests for materials should be addressed to B.A.S. (Email: brenda.schulman@stjude.org).

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