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The dynamic disulphide relay of quiescin sulphydryl oxidase

Nature volume 488, pages 414–418 (2012)Cite this article

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Abstract

Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins1,2. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids3 and has attracted attention owing to its overproduction in tumours4,5. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.

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Figure 1: TbQSOX undergoes domain reorientation to accomplish disulphide relay.
Figure 2: Changes in the TbQSOX conformational ensemble during catalysis.
Figure 3: Mammalian QSOX and mechanistic insights into the QSOX catalytic cycle.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the TbQSOX, TbQSOXC and HsQSOX1(PDI) structures have been deposited with the Protein Data Bank under accession codes 3QCP, 3QD9 and 3Q6O, respectively. The MmQSOX1C coordinates and structure factors have been deposited with accession codes 3T58 (orthorhombic) and 3T59 (monoclinic).

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Acknowledgements

S. Rogotner and O. Dym helped in growing the TbQSOXC crystals. We thank T. Ilani and A. Horovitz for reading of the manuscript and N. Nelson and his research group for help with X-ray data collection. A. Moseri assisted with high-performance liquid chromatography. This study was funded by the Israel Science Foundation. D.F. and A.A. acknowledge the Kimmelman Center for Macromolecular Assemblies for additional support. C.T. and V.K.K. acknowledge National Institutes of Health (NIH) grant GM26643. The molecular movie was produced using the University of California San Francisco Chimera package from the Resource for Biocomputing, Visualization and Informatics (supported by NIH grant P41 RR001081).

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Authors and Affiliations

  1. Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel,

    Assaf Alon, Iris Grossman, Yair Gat & Deborah Fass

  2. Department of Chemistry and Biochemistry, University of Delaware, Newark, 19716, Delaware, USA

    Vamsi K. Kodali & Colin Thorpe

  3. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA

    Frank DiMaio & David Baker

  4. Department of Biological Research Support, Weizmann Institute of Science, Rehovot 76100, Israel,

    Tevie Mehlman

  5. Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel,

    Gilad Haran

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  1. Assaf Alon
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Contributions

A.A. designed experiments, expressed, purified and crystallized proteins, and, together with D.F., solved and refined the TbQSOX, TbQSOXC and MmQSOX1C structures. A.A. also performed the FRET and crosslinking experiments. I.G. improved the TbQSOX crystals. Y.G. grew the MmQSOX1C crystals. V.K.K. and C.T. provided plasmids and helped to design and analyse experiments. F.D. and D.B. accomplished the molecular replacements to solve the HsQSOX1(PDI) structure. T.M. performed the mass spectrometry experiments and analyses. G.H. helped to design and analyse the FRET experiments and assisted with operation of the fluorimeter. D.F. expressed proteins, performed oxygen consumption measurements and designed and analysed the experiments. A.A. and D.F. wrote the manuscript.

Corresponding author

Correspondence to Deborah Fass.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14, which show experiments that demonstrate the quality of the protein preparations used in this study together with figures that depict in-depth analysis of features of the crystal structures and Supplementary Tables 1-5, which contain statistics and information about crystallography and mass spectrometry analysis. This file also contains a Supplementary Discussion, Supplementary References, and a legend for Supplementary Movie 1. (PDF 11421 kb)

Supplementary Movie 1

This movie depicts one hypothetical trajectory between the two crystallized conformations of Trypanosoma brucei QSOX. The starting conformation is the structure of wild-type TbQSOX; the target conformation is the structure of TbQSOXC, a variant containing a disulfide between the two redox-active domains. (MOV 18617 kb)

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Alon, A., Grossman, I., Gat, Y. et al. The dynamic disulphide relay of quiescin sulphydryl oxidase. Nature 488, 414–418 (2012). https://doi.org/10.1038/nature11267

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