Saturation mutagenesis of a predicted ancestral Syk‐family kinase

Abstract

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta‐chain‐associated protein kinase of 70 kDa [ZAP‐70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP‐70. Taking advantage of the ability to express this novel Syk‐family kinase in bacteria, we developed a two‐hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site‐saturation mutagenesis library of the kinase domain of this reconstructed Syk‐family kinase. Sites of loss‐of‐function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk‐family variant increases the soluble expression of the protein by 75‐fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk‐family kinase variants that can be expressed in bacteria with very high yield.

Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function

“…Furthermore, Rheostat scores (i) provide a convenient summary of the SSM datasets that are usually shown as large heatmaps ( e.g. , ( 20 , 21 , 22 , 23 , 24 , 25 )) but are difficult to digest or compare to structures, yet (ii) retain the position-specific information that is obscured in histograms that aggregate all substitution data for all positions ( e.g. , ( 23 , 25 , 26 , 27 )).…”