Abstract
Rhomboid proteases reside within cellular membranes, but the advantage of this unusual environment is unclear. We discovered membrane immersion allows substrates to be identified in a fundamentally-different way, based initially upon exposing 'masked' conformational dynamics of transmembrane segments rather than sequence-specific binding. EPR and CD spectroscopy revealed that the membrane restrains rhomboid gate and substrate conformation to limit proteolysis. True substrates evolved intrinsically-unstable transmembrane helices that both become unstructured when not supported by the membrane, and facilitate partitioning into the hydrophilic, active-site environment. Accordingly, manipulating substrate and gate dynamics in living cells shifted cleavage sites in a manner incompatible with extended sequence binding, but correlated with a membrane-and-helix-exit propensity scale. Moreover, cleavage of diverse non-substrates was provoked by single-residue changes that destabilize transmembrane helices. Membrane immersion thus bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics.DOI:http://dx.doi.org/10.7554/eLife.00173.001.
Keywords:
D. melanogaster; E. coli; Human; intramembrane proteolysis; pathogen; rhomboid protease.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Catalytic Domain
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Cell Membrane / chemistry*
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Cell Membrane / metabolism
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Drosophila Proteins / chemistry*
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism
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Drosophila melanogaster / chemistry
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Drosophila melanogaster / enzymology*
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Epidermal Growth Factor / chemistry*
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Epidermal Growth Factor / genetics
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Epidermal Growth Factor / metabolism
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Gene Expression
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Humans
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Hydrophobic and Hydrophilic Interactions
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Molecular Dynamics Simulation
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Molecular Sequence Data
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Mutation
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Protein Isoforms / chemistry
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Transport
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Proteolipids / chemistry
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Proteolipids / metabolism
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Proteolysis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Serine Endopeptidases
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Serine Proteases / chemistry*
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Serine Proteases / genetics
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Serine Proteases / metabolism
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Substrate Specificity
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rho GTP-Binding Proteins / chemistry*
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rho GTP-Binding Proteins / genetics
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rho GTP-Binding Proteins / metabolism
Substances
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Drosophila Proteins
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Membrane Proteins
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Protein Isoforms
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Proteolipids
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Recombinant Proteins
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proteoliposomes
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spi protein, Drosophila
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Epidermal Growth Factor
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Serine Proteases
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Serine Endopeptidases
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RHBDL2 protein, human
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Rho1 protein, Drosophila
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rho GTP-Binding Proteins