Tubulin Tyrosine Ligase-Mediated Modification of Proteins.

Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.

Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates.

Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.