Engineering a Tunnel to a More Versatile Lipoic Acid Ligase

Abstract

Protein‐polymer conjugates have become a rapidly expanding area of research. As a result of broad utility in a range of disciplines, protein‐polymer conjugates can now be applied to therapeutics, biofuels, and even protein tags. Grafting‐to and Grafting‐from methods have been successful in creating these conjugates but have limitations on the specificity of attachment (Lele et al. 2005). Lipoic acid ligase (LplA) has been demonstrated to be convenient tool for attaching small molecule precursors that can enable subsequent attachment of a polymer to specific sites of target proteins containing a lipoic acid ligase acceptor peptide (LAP) sequence. Studies on LplA have demonstrated that mutation of tryptophan 37 to valine allows LplA to accommodate 10‐azidodecanoic acid (Plaks et al., 2015). Paired with an alkyne‐bearing polymer this enables the use of click chemistry to attach a polymer to the protein‐ligated 10‐azidodecanoic acid. While useful, attachment of a pre‐clicked polymer would enable for more rapid exploration of protein‐polymer conjugates with a wide library of polymers. In this study, we describe structure‐guided, activity‐based efforts to engineer a tunnel within LplA to further open the binding pocket and allow LplA to accept pre‐clicked polymers.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).