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EMBO J. 2014 Oct 16;33(20):2408-21. doi: 10.15252/embj.201489367. Epub 2014 Sep 12.

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures.

Author information

1
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
2
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic Department of Microbiology, Faculty of Science, Charles University, Prague, Czech Republic.
3
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
4
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic kvido.strisovsky@uochb.cas.cz.

Abstract

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.

KEYWORDS:

intramembrane protease; rhomboid family; rhomboid protease; structure; substrate recognition

PMID:
25216680
PMCID:
PMC4253528
DOI:
10.15252/embj.201489367
[Indexed for MEDLINE]
Free PMC Article

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