Membrane enzymes 'stop and frisk' proteins indiscriminately
In a fascinating new study, researchers report on rhomboid proteases, a class of enzymes present in almost all species whose active site is buried in the lipid bilayer, leading them to cleave other membrane proteins within their transmembrane domains.
How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown.
Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes.
This new finding that an enzyme shows no affinity (“or little, if any meaningful affinity”) for its substrate is remarkable — enzymes are usually thermodynamically controlled, not kinetically.
Naturally the authors went out of their way to ensure this matched in vivo conditions. After having done so with a host of different biochemical techniques including FRET (schematic shown below), they examined proteolysis in detergent micelles (artificial membranes) with another neat technique called equilibrium gel filtration, more common in pharmacological studies, with which Kd can accurately be measured.
The “subset of DNA repair enzymes” alluded to in the excerpt above are DNA glycosylases, which catalyse the first step in base excision repair (aka AP [apurinic/apyrimidinic] repair, where a base pair is missing on the DNA).
DNA glycosylases remove damaged bases from DNA using an intriguing mechanism that involves two different enzyme sites. Nucleotides flipped out of a DNA double helix first interact with an “interrogation site” on the DNA glycosylase. Importantly, a damaged base is not bound with high affinity per se; instead, it is able to spend more time in the dynamic, extrahelical state and thus stay longer in the interrogation complex. This longer residence allows the base to translocate to a second, deeper site—the excision site—where the glycosidic linkage is clipped to excise the base from DNA. The discriminatory mechanism is therefore rate governed, with a minor contribution from binding affinity to the damaged base itself. The second key property of these DNA glycosylases is a slow kcat because it ensures that catalysis is slower than the residence time of natural bases, kinetically protecting them from hydrolysis.
These striking parallels suggest that low substrate affinity and slow rate of rhomboid proteolysis are not defects but, rather, features of this enzyme system.
The group hypothesise that perhaps rhomboid proteases also use an analagous “interrogation” site to kinetically discriminate substrate from otherwise. The suggestion they outline below is portrayed in the diagram above for a helical domain (red).
Although the gate has been viewed simply as a point of substrate entry, the crevice created by gate opening, which is stable in the membrane, may actually be an “interrogation” site. Like with DNA glycosylases, this site is physically separated from the deeper active site, which would force transmembrane helices to reside in the unwound state to reach the catalytic residues for proteolysis to ensue, instead of returning laterally to the membrane. Our recent spectroscopy analysis of substrates revealed that they form inherently less-stable helices than nonsubstrates, which suggests that they would spend more time in the unfolded state and thus reach the inner “scission site” from the “interrogation site.”..
It is thus tempting to speculate that the primordial function of rhomboid proteases was to patrol the membrane looking for unfolded membrane proteins to cleave as a repair mechanism analogous to how DNA glycosylases patrol the genome for damaged bases.
Rhomboid proteases resemble water-filled barrels with side gates for protein entry, as shown in the main image, above. This work suggests that while both stable and unstable proteins enter the side gate, stable proteins likely remain intact and drift back out into the membrane in the 2 and a half minutes it takes the protease to strike.
Unstable proteins however will start to wobble in the watery interior (giving away their instability, previously masked by the stabilising, viscous membrane) and struggle to exit the enzyme's “barrel” — hence they will get snipped and recycled.
While the authors remain cautious, the existence of iRhoms (not mentioned in the piece), pseudoproteases that instead of cleaving proteins, hijack the ER-assisted degradation (ERAD) pathway to release them for proteasomal destruction, suggests another level of control if indeed this is (or was evolutionarily) a repair mechanism.
Whether weak binding at transmembrane sites is important for catalysis inside the membrane, or a deliberate specialization by this class of enzymes, remains to be determined.
Dickey et al. (2013) Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity. Cell, 155(6) 1270-81
