Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins.

Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates.

Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection.

The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.6 µs) and with bound Ca2+ (simulations of 1.6 µs, 1.8 µs, and 2.5 µs). Our results show that Ca2+ binding causes not only ordering of the disordered ß6/ß7 loop of the protein, but also modulates hinge motions in the dynamic ß7/ß8 loop, which is important for the catalytic activity of the enzyme. Cation binding triggers signal transmission from the Ca2+ binding site to the dynamic ß7/ß8 loop via the repetitive folding/unfolding of short helical stretches of the disordered ß6/ß7 loop. These correlated structural rearrangements lead to several distinct conformational states of the binding groove, which show optimal binding features for the sorting signal motif and feature binding energies up to 20 kcal/mol more favorable than observed for the sortase A without Ca2+. The presented results indicate a highly correlated, conformational selection-based activation mechanism of the enzyme triggered by cation binding. They also demonstrate the importance of the dynamics of intrinsically disordered regions for allosteric regulation.