Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Abstract

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

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

“…TAMP inhibitory activity and glutamine accessibility for MTG are obviously counter‐regulated functions of SSTI and raise the question of how a more extended variant may influence SSTI. The result confirmed that MTG‐reactive glutamines are preferably located in flexible protein regions, especially at the terminal ends . MTG‐mediated side‐chain hydrolysis, however, is most likely not the reason for loss of reactive glutamines in the course of S. mobaraensis growth as hypothesized earlier , but successive removal of the N‐terminal extension peptide.…”

“…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).…”

Efficient Site‐Specific Antibody–Drug Conjugation by Engineering a Nature‐Derived Recognition Tag for Microbial Transglutaminase

“…Apart from the evident success of mTG applications in diverse fields, the physiological role of this enzyme in its host S. mobaraensis is barely understood. Nevertheless, over the past decades the reactivity of glutamine sites within intrinsic substrate proteins have been thoroughly investigated . Our recent research revealed a novel recognition sequence for microbial transglutaminase that was derived from its natural substrate dispase autolysis‐inducing protein (DAIP).…”