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Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

Nature Structural & Molecular Biology volume 17, pages 1352–1357 (2010)Cite this article

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Abstract

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

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Figure 1: Mpa1–234 hexamer has three 75 Å–long coiled coils that are needed for Pup recognition.
Figure 2: Full-length Pup in the context of hexameric Mpa1–234.
Figure 3: The Pup helical region is essential for proteasomal degradation, which supports the idea that Mpa uses a binding-induced folding recognition mechanism.

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Acknowledgements

We thank K. Burns for reviewing this manuscript and C. Nathan for advice and encouragement. X-ray diffraction data for this study were collected at beamlines X25 and X29 of the National Synchrotron Light Source. Financial support was principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the US National Institutes of Health (NIH). This work was supported by NIH grant AI070285 and Brookhaven National Laboratory LDRD grant 10-016 to H.L. and by NIH grant HL092774 to K.H.D. K.H.D. was also supported by a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases award.

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Authors and Affiliations

  1. Biology Department, Brookhaven National Laboratory, Upton, New York, USA

    Tao Wang & Huilin Li

  2. Department of Microbiology, New York University School of Medicine, New York, New York, USA

    K Heran Darwin

  3. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Huilin Li

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  1. Tao Wang
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Contributions

T.W. performed protein purification, crystallization and structure determination; K.H.D. performed mutagenesis and in vivo degradation assays; T.W., K.H.D. and H.L. designed experiments and wrote the manuscript.

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Correspondence to K Heran Darwin or Huilin Li.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 2054 kb)

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Wang, T., Darwin, K. & Li, H. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation. Nat Struct Mol Biol 17, 1352–1357 (2010). https://doi.org/10.1038/nsmb.1918

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