Domain Swapping in the Cytoplasmic Domain of the Escherichia coli Rhomboid Protease

Rhomboids are membrane-embedded serine proteases that cleave membrane protein substrates. Escherichia coli rhomboid GlpG (ecGlpG) consists of an N-terminal cytoplasmic domain and a membrane domain containing the active site. We determined the crystal structure of the soluble cytoplasmic domain of ecGlpG at 1.35 Å resolution and examined whether this domain affected the catalytic activity of the enzyme. The structure revealed that the ecGlpG cytoplasmic domain exists as a dimer with extensive domain swapping between the two monomers. Domain-swapped dimers can be isolated from the full-length protein, suggesting that this is a physiologically relevant structure. An extensive steady-state kinetic analysis of the full-length ecGlpG and its membrane domain using soluble and transmembrane model protein substrates resulted in an unexpected conclusion: removal of the cytoplasmic domain does not alter the catalytic parameters for detergent-solubilized rhomboid for both substrates.

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► Domain swapping exists in the crystal structure for the cytoplasmic domain of ecGlpG.
► Both monomeric and dimeric forms of the cytoplasmic domain exist in solution.
► ecGlpG catalytic parameters (Km, Vmax, and kcat) were determined with soluble and transmembrane substrates.
► The cytoplasmic domain of ecGlpG does not affect enzyme activity with model substrates.