BTK autoinhibition analyzed by high-throughput swaps of SH2 domains

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src-family kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, stabilizing the inhibitory conformation of the SH3 domain; in Abl, interaction between the kinase domain and a myristoyl group on the N-terminal segment provides a similar latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an obvious SH2-kinase latch. To assess the role of the SH2 domain in autoinhibition of BTK, we generated hundreds of chimeric BTK molecules in which the native SH2 domain is replaced by other SH2 domains. We measured the fitness of these chimeric proteins using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis ..., Quantification of fitness from sequencing data was performed as in Eisen et al., Sci Signal. 2024. Briefly, Fastq files from MiSeq runs were aligned to the Fasta files containing the full sequences of each variant using Kallisto (Bray, Nat. Biotech. 2016) to generate read counts for each variant. A read cutoff of 50 reads was applied to the input libraries such that any variant not passing this threshold was discarded. Next, the unnormalized scores were calculated by dividing the number of reads in the sorted dataset by the number of reads in the input dataset and taking the log10. These unnormalized scores were normalized by subtracting the mean of the wild-type fitness scores. The SH2-domain library included 22 synonymous wild type sequences that were generated by randomly choosing 5 codons and substituting them for randomly chosen synonymous counterparts. Sequences that introduced additional BsaI restriction sites were avoided. In the helix I library, 44 synonymous wild type seq..., , # BTK autoinhibition analyzed by high-throughput swaps of SH2 domains

[https://doi.org/10.5061/dryad.rxwdbrvjx](https://doi.org/10.5061/dryad.rxwdbrvjx)

## Description of the data and file structure

These datasets comprise RNA-seq data that quantify fitness scores for BTK or variants. One set of data (SH2) swaps 328 SH2 domains into the BTK protein. Another set (Helix I) swaps the C-terminal helix in the BTK kinase domain for 118 Tec-kinase C-terminal helices from jawed vertebrates. The third set (Abundance) measures the protein abundance of each SH2 chimera using fluorescently tagged variants of the proteins and cell sorting. Each replicate has an input (the denominator in the fitness calculation) and a sort sample (the CD69-selected reads, or the numerator in the calculation). In addition, the SH2 and Helix I experiments were performed in two human lymphocyte cell lines: Jurkat T cells and Ramos B cells that had been knocked out for ITK and BTK, respectively. The abundance measurem...,