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.
Proteases are enzymes that break the peptide bonds that hold proteins together, and have a central role in many physiological processes, including digestion, blood clotting and programmed cell death. An important characteristic of proteases is that they are highly selective, only cutting proteins that contain well-defined sequences of amino acids in accessible regions. Proteases that are soluble in water have been studied for over a century and are now well understood, as are proteases that need to be tethered to the membrane of a cell to work properly.
In 1997 researchers discovered a protease that was immersed in the cell membrane, and it soon became clear that these intramembrane proteases were widespread and involved in a wide range of processes in cells. Examples of intramembrane proteases include γ-secretase, which is implicated in Alzheimer's disease, and various site-2 proteases that regulate pathogenic circuits in bacteria.
There are many similarities between soluble and intramembrane proteases. However, given that intramembrane proteases evolved within the hydrophobic environment of the membrane, whereas soluble proteases evolved in an aqueous environment, there should there should also be significant differences between them. The best understood intramembrane proteases in terms of their biochemistry are probably the rhomboid proteases. However, most studies of their function have been performed in detergent systems rather than in real membranes.
Moin and Urban now report that the main strategy used by rhomboid proteases to identity the proteins that they selectively cut is completely different from that used by soluble proteases. Through a combination of biochemical and spectroscopic methods, they have discovered that rhomboid proteases identify the proteins they act on mainly by detecting changes in dynamic behavior: only those proteins that lose a stable helical structure when they exit the lipid phase to interact with the rhomboid protease will be cut by the rhomboid protease. Soluble proteases, on the other hand, achieve specificity by looking for proteins with a particular sequence of amino acids. The novel strategy used by rhomboid proteases allows them to patrol the membrane for unstable helices and selectively cut them. This discovery provides the first explanation of why these complicated enzymes evolved to have active sites immersed within the cell membrane.