Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing

Abstract

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel positive selections combined with deep sequencing and statistical analysis and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNA pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure determination and synthetic protein kinase activation.

Chemical biology approaches for studying posttranslational modifications

“…Recently, multi-directional efforts for biosynthesis of phosphothreonine and evolution of a pThrRS/tRNA pair via parallel selections with deep sequencing led to an efficient genetic encoding of phosphothreonine. 51 The utility of this system was demonstrated by producing catalytically active protein kinase Cdk2 installed with phosphothreonine at position 160.…”

Conserved phosphorylation hotspots in eukaryotic protein domain families

“…These studies are typically difficult to perform since it is not straightforward to obtain large amounts of purified phospho-protein, in particular in a residue specific manner. However, recent progress in genetically encoded phosphorylated residues in protein expression systems (Rogerson et al, 2015;Zhang et al, 2017) should make these studies more feasible. Such studies can in turn spur the development of rational design of phosphorylation switches.…”

Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

“…It also offers the freedom of incorporating an UAA at any position of the target protein, not restricted to the C terminus as in the EPL reaction. A variety of UAAs bearing PTM functionalities has been incorporated, such as phospho-Ser, Thr, and Tyr (Park et al, 2011;Lee et al, 2013;George et al, 2016;Hoppmann et al, 2017;Luo et al, 2017;Zhang et al, 2017a); acetylated Lys (Neumann et al, 2008); and methylated Lys (Nguyen et al, 2009(Nguyen et al, , 2010Ai et al, 2010;Groff et al, 2010;Wang et al, 2017b). Furthermore, a Lys analog with a d-thiol entity is also available for incorporation as an UAA, opening the possibility of synthesizing UB chains of various linkages with the combined use of UAA incorporation and EPL ( Fig.…”