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Ten catalytic snapshots of rhomboid intramembrane proteolysis from gate opening to peptide release

Nature Structural & Molecular Biology volume 26, pages 910–918 (2019)Cite this article

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Abstract

Protein cleavage inside the cell membrane triggers various pathophysiological signaling pathways, but the mechanism of catalysis is poorly understood. We solved ten structures of the Escherichia coli rhomboid protease in a bicelle membrane undergoing time-resolved steps that encompass the entire proteolytic reaction on a transmembrane substrate and an aldehyde inhibitor. Extensive gate opening accompanied substrate, but not inhibitor, binding, revealing that substrates and inhibitors take different paths to the active site. Catalysis unexpectedly commenced with, and was guided through subsequent catalytic steps by, motions of an extracellular loop, with local contributions from active site residues. We even captured the elusive tetrahedral intermediate that is uncleaved but covalently attached to the catalytic serine, about which the substrate was forced to bend dramatically. This unexpectedly stable intermediate indicates rhomboid catalysis uses an unprecedented reaction coordinate that may involve mechanically stressing the peptide bond, and could be selectively targeted by inhibitors.

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Fig. 1: Core chemical mechanism of serine protease catalysis.
Fig. 2: Comparison of precatalytic GlpG conformers.
Fig. 3: Time-resolved X-ray crystallography of aldehyde inhibitor binding and catalysis.
Fig. 4: Time-resolved X-ray crystallography of transmembrane substrate binding and cleavage.
Fig. 5: Enzyme analysis validates a catalytic role for the L5 loop in intramembrane proteolysis.
Fig. 6: Model of enzymatic actions underlying rhomboid intramembrane proteolysis.

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Data availability

Coordinates of all structures have been deposited into the Protein Data Bank under accession codes 6PJ4 (Snapshot-I1), 6PJ5 (Snapshot-I2), 6PJ7 (Snapshot-I3), 6PJ8 (Snapshot-I4), 6PJ9 (Snapshot-S1), 6PJA (Snapshot-S2), 6PJP (Snapshot-S3), 6PJQ (Snapshot-S4), 6PJR (Snapshot-S5) and 6PJU (Snapshot-S6). Source data for Fig. 5b–d are available with the paper online. Any other data are available from the authors upon reasonable request.

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Acknowledgements

We are grateful to current and past members of the Urban laboratory for discussions and support, as well as the expert, friendly and helpful staff at the Cornell High Energy Synchrotron Source (CHESS) for access to beam time. This work was supported by a National Institutes of Health grant (no. R01AI066025 to S.U.). All X-ray diffraction data were collected using instruments at CHESS (beamline supported by National Science Foundation grant no. DMR-0936384 and National Institutes of Health grant no. GM-103485).

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  1. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Sangwoo Cho, Rosanna P. Baker, Ming Ji & Siniša Urban

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Contributions

S.U. and S.C. conceived the research. S.U. made all DNA constructs. S.C. performed protein purification, X-ray crystallography and structure determination. R.P.B. performed protein purification, enzyme kinetics and thermostability analyses. M.J. conducted EPR spectroscopy. S.U. wrote the paper and all authors approved the final version of the manuscript.

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Correspondence to Siniša Urban.

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Peer review information Ines Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figure 1 Time-resolved X-ray crystallographic analysis of aldehyde inhibitor binding and catalysis.

A simplified schematic of GlpG action on a peptide aldehyde (see primary Fig. 3 for stereoimages and electron densities). Waters appear as red spheres, while the L5 loop is colored in pink throughout for emphasis. To obtain these structural snapshots, crystals of GlpG in a bicelle membrane were soaked with the Ac-VRMA-CHO peptide aldehyde for increasing lengths of time prior to freezing and X-ray diffraction analysis. In addition to the catalytic steps, the interactions that formed with the L5 loop are detailed in the lower left image (panel c), which is the same data as in panel b but includes L5 sidechain/mainchain interaction details.

Supplementary Figure 2 Time-resolved X-ray crystallographic analysis of transmembrane substrate binding and cleavage.

A simplified schematic of catalytic action by GlpG on a peptide substrate (see primary Fig. 4 for stereoimages and electron densities). To obtain these structural snapshots, crystals of GlpG in a bicelle membrane were soaked with the Ac-RKVRMAAIVFSFP-amide peptide for increasing lengths of time prior to freezing and X-ray diffraction analysis. The abrupt substrate bend is detailed in the lower left image, which is the same data as the panel above it but rotated ~90° to the right. Waters are rendered as red spheres, while disordered regions are highlighted in red (in b, e, f, h) or grey (in d) during the reaction steps.

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Cho, S., Baker, R.P., Ji, M. et al. Ten catalytic snapshots of rhomboid intramembrane proteolysis from gate opening to peptide release. Nat Struct Mol Biol 26, 910–918 (2019). https://doi.org/10.1038/s41594-019-0296-9

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