TU Darmstadt / ULB / TUbiblio

The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.

Juettner, Norbert E. and Schmelz, Stefan and Anderl, Anita and Colin, Felix and Classen, Moritz and Pfeifer, Felicitas and Scrima, Andrea and Fuchsbauer, Hans-Lothar (2019):
The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.
In: The FEBS journal, (ePub ahead of Print), ISSN 1742-4658,
DOI: 10.1111/febs.15044,
[Article]

Abstract

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, i. e. determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues, respectively. Structural integrity of the mutants was verified by determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analyzed, which superposes well with SSI-family proteins. This article is protected by copyright. All rights reserved.

Item Type: Article
Erschienen: 2019
Creators: Juettner, Norbert E. and Schmelz, Stefan and Anderl, Anita and Colin, Felix and Classen, Moritz and Pfeifer, Felicitas and Scrima, Andrea and Fuchsbauer, Hans-Lothar
Title: The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.
Language: English
Abstract:

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, i. e. determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues, respectively. Structural integrity of the mutants was verified by determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analyzed, which superposes well with SSI-family proteins. This article is protected by copyright. All rights reserved.

Journal or Publication Title: The FEBS journal
Number: ePub ahead of Print
Divisions: 10 Department of Biology
10 Department of Biology > Microbiology and Archaea
Date Deposited: 26 Aug 2019 11:07
DOI: 10.1111/febs.15044
Identification Number: pmid:31420998
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)

View Item View Item