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Hybrid molecular structure of the giant protease tripeptidyl peptidase II

Nature Structural & Molecular Biology volume 17, pages 990–996 (2010)Cite this article

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Abstract

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo–electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.

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Figure 1: Crystal structure of the TPP II dimer.
Figure 2: Stereo view of the substrate-binding cleft.
Figure 3: Active-site region of subtilisin and TPP II dimer.
Figure 4: Hybrid model of the TPP II spindle.
Figure 5: Density at the dimer-dimer contact region and the relocation of loop L2.
Figure 6: Chamber system of TPP II and pathways to the active sites.
Figure 7: Schematic diagram of the proposed assembly-dependent activation mechanism.

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Acknowledgements

Diffraction datasets for the structure determination were collected at Beamline 8.2.2, Advanced Light Source, Lawrence Berkeley National Laboratory. We would like to thank C. Ralston and her beamline staff for their assistance. We thank A. Sonnen for the calculation of the contact areas. This work is supported by funding from the Deutsche Forschungsgemeinschaft (B.R.), the National Institutes of Health (B.K.J.) and the US Department of Energy.

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Author notes

  1. Crystal K Chuang and Beate Rockel: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Crystal K Chuang, Peter J Walian & Bing K Jap

  2. Graduate Group in Comparative Biochemistry, University of California, Berkeley, California, USA

    Crystal K Chuang & Bing K Jap

  3. Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

    Beate Rockel, Gönül Seyit, Anne-Marie Schönegge, Jürgen Peters & Wolfgang Baumeister

  4. Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Petrus H Zwart

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  1. Crystal K Chuang
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Contributions

W.B. and B.K.J. conceived the project; X-ray crystallography work, from purification to final structure, was conducted by C.K.C. under the guidance of B.K.J.; P.J.W. was involved in data collection and processing and P.H.Z. in initial processing of data; G.S. and J.P. were involved with initial purification and crystallization trials; electron microscopy structural studies were performed by B.R.; A.-M.S. and J.P. performed mutation and functional studies; the manuscript was written by C.K.C., B.R., J.P., B.K.J. and W.B.; All authors read and edited the manuscript.

Corresponding authors

Correspondence to Wolfgang Baumeister or Bing K Jap.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, Supplementary Methods (PDF 8659 kb)

Supplementary Video 1

Rotation about the spindle axis of two dimers docked into the central part of the TPP II envelope. The color–coding for the dimers is the same as used for Figure 1: yellow, subtilisin domain (residues 1–522); red, active site residues (Asp44, His272, Ser462, Asn374); orange, insert (residues 75–266); green, central domain (residues 523–1098); blue, C–terminal domain (residues 1099–1354); the adjoining monomers are shown in a lighter shade of the same color scheme. (MOV 3620 kb)

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Chuang, C., Rockel, B., Seyit, G. et al. Hybrid molecular structure of the giant protease tripeptidyl peptidase II. Nat Struct Mol Biol 17, 990–996 (2010). https://doi.org/10.1038/nsmb.1870

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