Allosteric regulation of rhomboid intramembrane proteolysis

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)‐based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii ). We demonstrate significant differences in catalytic efficiency ( k cat / K 0.5 ) values for transmembrane substrate psTatA (TatA from Providencia stuartii ) cleavage for three rhomboids: AarA from P. stuartii , ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite‐mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. Synopsis Intermembrane proteases of the rhomboid family, thought to have broad substrate specificity and to only partly rely on cleavage recognition motifs in substrates, exhibit considerable substrate specificity due to positive cooperativity and allosteric substrate activation. Rhomboids specifically recognize their substrates in gel‐ and FRET‐based kinetic assays Rhomboids cleave the TatA substrate with positive cooperativity Recognition of TatA substrate by its physiological rhomboid protease AarA involves an exosite Dimerization is important for transmembrane substrate cleavage Soluble model substrate casein can be cleaved by dimers and monomers in a non‐cooperative manner Graphical Abstract Homotropic allosteric communication between rhomboid dimers controls efficient cleavage of cognate transmembrane substrate while being dispensable for proteolysis of generic soluble substrates.