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Architectural and thermodynamic principles underlying intramembrane protease function

Nature Chemical Biology volume 8, pages 759–768 (2012)Cite this article

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Abstract

Intramembrane proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid proteases maintain low intrinsic thermodynamic stability (ΔG = 2.1–4.5 kcal mol−1) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution.

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Figure 1: Thermodynamic assessment of rhomboid protease stability.
Figure 2: Differential static light scattering as a probe of rhomboid stability.
Figure 3: Only two regions of hydrogen bonding are critical for GlpG stability.
Figure 4: A small number of van der Waals interactions are critical for GlpG architecture and catalysis.
Figure 5: Quantitative assessment of peripheral TM interactions and catalytic residues: implications for dynamics.
Figure 6: Thermodynamic assessment of GlpG architectural mutants.
Figure 7: Natural diversity in the thermostability of rhomboid proteases.
Figure 8: Architectural principles underlying rhomboid protease function.

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Acknowledgements

We are grateful to all members of the Urban lab and to D. Otzen for stimulating scientific discussions and to the Malaria Research Institute Biophysics Core for use of their CD spectropolarimeter. This work was supported by the Howard Hughes Medical Institute and the David and Lucile Packard Foundation.

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  1. Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Rosanna P Baker & Sinisa Urban

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Contributions

R.P.B. designed research, performed all biochemical experiments (except CD spectroscopy), analyzed data and prepared all figures; S.U. designed research, made all DNA constructs, performed CD spectroscopy, analyzed the data and wrote the paper.

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Correspondence to Sinisa Urban.

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Baker, R., Urban, S. Architectural and thermodynamic principles underlying intramembrane protease function. Nat Chem Biol 8, 759–768 (2012). https://doi.org/10.1038/nchembio.1021

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