Reporter Immobilization Assay (REIA) for Bioconjugating Reactions.

Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 µg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour.

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.