Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase

Abstract

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

Structure of a glutamine donor mimicking inhibitory peptide shaped by the catalytic cleft of microbial transglutaminase

“…The attack of lysine‐containing donor proteins or other primary amines that enter the active site from the rear vestibule then provides opportunities for protein cross‐linking via γ‐glutamyl ε‐lysine isopeptide bonds or glutamine transamidation under release of the enzyme. Although great efforts have been made to rationalize specific selection of the most abundantly modified endo ‐glutamines by MTG , mechanisms that lead to attraction of substrate proteins by long‐range forces and that determine the orientation remained largely elusive. For instance, S. mobaraensis secretes, beside MTG, the Dispase‐autolysis inducing protein (DAIP, UniProt http://www.uniprot.org/uniprot/P84908, PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FZP) clustering four out of five glutamines in a seven‐bladed β‐propeller fold .…”

“…Although great efforts have been made to rationalize specific selection of the most abundantly modified endo ‐glutamines by MTG , mechanisms that lead to attraction of substrate proteins by long‐range forces and that determine the orientation remained largely elusive. For instance, S. mobaraensis secretes, beside MTG, the Dispase‐autolysis inducing protein (DAIP, UniProt http://www.uniprot.org/uniprot/P84908, PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FZP) clustering four out of five glutamines in a seven‐bladed β‐propeller fold . The glutamine of an extended loop, Gln39, has been shown to be the preferred binding site of MTG.…”

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

“…Contrary to SSI and many SIL proteins, SSTI contains two adjacent glutamine residues that represent the most probable glutamine donor sites for cross‐linking by transglutaminase (MTG). Both residues, located in the SSTI extension peptide, obviously exhibit the required degrees of freedom to enter the MTG active core .…”

“…Glutamine accessibility of MTG was determined by enzymatic conjugation of SSTI with biotin cadaverine (5‐biotinylamidopentylamine) as previously described . The biotinylated proteins were separated by electrophoresis, blotted and visualized by fluorescent streptavidin IRDye 800 CW conjugates.…”

Biochemical study of sortase E2 from Streptomyces mobaraensis and determination of transglutaminase cross‐linking sites

“…While failure of Sm-MTG to label Gln-129 and Gln-176 may be the result of backbone interaction and the occurrence of charged and bulky residues in close vicinity [21,40,41], the access to Gln-62 and Gln-65 was most likely caused by a largely disordered N-terminal D 1-50 -Sm-SrtE2 peptide (Table S1). While failure of Sm-MTG to label Gln-129 and Gln-176 may be the result of backbone interaction and the occurrence of charged and bulky residues in close vicinity [21,40,41], the access to Gln-62 and Gln-65 was most likely caused by a largely disordered N-terminal D 1-50 -Sm-SrtE2 peptide (Table S1).…”