Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate

Abstract

Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure-function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4-to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.cell signaling ͉ presenilin ͉ signal peptide peptidase ͉ site-2 protease

Large Lateral Movement of Transmembrane Helix S5 Is Not Required for Substrate Access to the Active Site of Rhomboid Intramembrane Protease

“…Combining the two single mutations did not produce any further activity enhancement. The 2-fold increase in the double mutant is in general agreement with the published study (22), which documented a 7-fold activity enhancement.…”

“…To examine whether S5 undergoes any major conformational change during the binding and hydrolysis of real protein substrate, we studied the effect of cross-linking S5 to a neighboring helix (S2) on the activity of the protease against a fusion protein containing the TM region of Gurken, a natural substrate of Drosophila rhomboids (31). This approach has been attempted in an earlier study, but the result was not conclusive (22). In the published study, S5 was cross-linked to S2 through disulfide bonds between pairs of cysteines introduced to their interface by mutagenesis (Y160C/L229C, W157C/ F232C, and F153C/W236C).…”

“…Mutations at the S2-S5 Interface-An earlier study (22) had identified two double mutants (F153A/W236A and W157A/ F232A) at the interface between TM helices S2 and S5 that significantly enhanced GlpG enzymatic activity. It was hypothesized that the mutations promoted gate opening by weakening the interaction between the two helices (Fig.…”

“…1B). This movement opens a gate within the membrane and allows substrate to enter laterally (16,22). Here, we examined the S5 gating model and critically evaluated published data that appear to support it.…”

Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

“…Reaction products were resolved and quantified by infrared fluorescence (LiCor Biosciences, Lincoln, NE) using western analysis (Baker et al, 2007). …”