Unraveling Synthetase's Mode of Action: The Pyrrolysyl‐tRNA Synthetase Dimer Uses Secondary Binding Sites in the Cell

Abstract

ABSTRACT Aminoacyl‐tRNA synthetases mediate the activation and transfer of amino acids to their cognate tRNA, which constitutes one of the initial events in protein biosynthesis. Even though different mechanisms of action have been proposed for the catalysis of these enzymes, their entire catalytic cycle remains elusive. Here, we used electron paramagnetic resonance spectroscopy in vitro and in cells in combination with molecular dynamics simulations to study the role of amino acid interactions in the catalytic cycle of pyrrolysyl‐tRNA synthetases (PylRS), a widely used tool for genetic code expansion. Experiments using the paramagnetic non‐canonical amino acid SLK‐1 revealed the presence and occupation of secondary amino acid binding sites in PylRS located at the intermonomer interface, distant from the catalytic binding site. Based on our results, we propose a model that assumes an alternating mode of action of the two PylRS monomers for the catalytic cycle of PylRS.