The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.
- MeSH
- chloromethylketony aminokyselin chemická syntéza farmakologie MeSH
- DNA vazebné proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- endopeptidasy chemie genetika metabolismus MeSH
- Escherichia coli chemie enzymologie genetika MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- membránové proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- molekulární modely * MeSH
- mutace MeSH
- proteiny z Escherichia coli antagonisté a inhibitory chemie genetika metabolismus MeSH
- Providencia chemie MeSH
- rekombinantní proteiny MeSH
- simulace molekulární dynamiky * MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
