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Active site remodelling accompanies thioester bond formation in the SUMO E1

Nature volume 463, pages 906–912 (2010)Cite this article

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Abstract

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 Å, respectively. These structures show that side chain contacts to ATP·Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

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Figure 1: Analogues of the Ub/Ubl-adenylate and E1∼Ub/Ubl tetrahedral intermediates.
Figure 2: Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediate.
Figure 3: Conformational changes within the Cys domain.
Figure 4: Active sites in E1/SUMO1-AMSN and E1∼SUMO1-AVSN.
Figure 5: Side chains required for adenylation are dispensable for formation of the tetrahedral intermediate analogue.
Figure 6: Side chains required for formation of the thioester bond or the tetrahedral intermediate analogue are dispensable for adenylation.

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The atomic coordinates and structure factors have been deposited in the RCSB database under accession codes 3KYC and 3KYD.

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Acknowledgements

We thank A. Armstrong for Ub and Ub E1 clones and N. Takeda for assistance in graphic art preparation. This work is based on research conducted at the NE-CAT beamlines of the Advanced Photon Source, supported by RR-15301 from the NCRR at the NIH. Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. D.S.T. is an Alfred P. Sloan Research Fellow. X.L. and D.S.T. were supported by NIH R01 AI068038 and the NYSTAR Watson Investigator Program. S.K.O., A.D.C. and C.D.L. were supported by NIH R01 GM065872, F32 GM075695 (A.D.C.), and the Rita Allen Foundation (C.D.L.).

Author Contributions Experiments were performed by S.K.O., A.D.C., X.L. and C.D.L. Data were analysed by S.K.O., A.D.C., X.L., D.S.T. and C.D.L. The manuscript was prepared by S.K.O., D.S.T. and C.D.L.

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  1. Structural Biology and,,

    Shaun K. Olsen, Allan D. Capili & Christopher D. Lima

  2. Molecular Pharmacology and Chemistry Programs, Sloan-Kettering Institute, New York, New York 10065, USA ,

    Xuequan Lu & Derek S. Tan

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  1. Shaun K. Olsen
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  2. Allan D. Capili
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Correspondence to Derek S. Tan or Christopher D. Lima.

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Olsen, S., Capili, A., Lu, X. et al. Active site remodelling accompanies thioester bond formation in the SUMO E1. Nature 463, 906–912 (2010). https://doi.org/10.1038/nature08765

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