​​Intramembrane protease specificity and catalytic mechanism.

They sound hard but they just became easier.

Intramembrane proteases regulate such activities as intercellular signaling, stress responses, and membrane protein quality control, which makes these widely conserved membrane proteins biologically very important. Since these unusual enzymes operate at the interface of hydrophobic lipid membrane and aqueous environment, the features of their catalytic mechanisms and regulation are distinct from the classical, water-soluble proteases. A true understanding of mechanisms of intramembrane proteases requires structural characterization of complexes with their transmembrane substrates. This is particularly challenging. 

Kvido Strisovsky and his team at the Institute of Organic Chemistry and Biochemistry (Academy of Sciences of the Czech Republic) had exactly this goal. They wanted to solve the structure of a substrate bound rhomboid protease GlpG with the hope that it would provide insights into the mechanism of action and the structural basis of substrate specificity. This goal presents inherent difficulties and challenges to overcome. The main 'trick' Dr. Strisovsky's group used was to make stable complexes of rhomboid protease with substrate-derived, mechanism-based inhibitors.  

In their paper “Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures” Dr. Strisovsky’s group presents structures which show the S1 to S4 subsites of the rhomboid protease, GlpG, involved in the binding of the substrate. The group went further and proposes that regions topologically homologous to the L1 loop of rhomboid protease could have similar substrate or client-protein binding function also in the wider rhomboid-like protein superfamily. The final concept introduced in the paper was a model of the Michaelis complex formed by a substrate bound in the active site of GlpG.  The paper, thus, provides the first direct structural view of rhomboid specificity and catalytic mechanism.

n-Dodecyl-β-D-Maltoside (DDM) was used for purifying the full length protein, whereas n-Decyl-β-D-Maltoside (DM) was used for stabilizing as well as purifying the core domain for crystallography.

 
For more information on Dr. Strisovsky’s work, please read “Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures in the EMBO Journal
(1)”.

 

Reference:
  1. Zoll, S., Stanchey, S., Began, J., Skerle, J., Lepsik, M., Peclinovska, L., Maier, P., and Strisovsky, K. (2014) EMBO J. 33(20), 2408-2421.

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