Go big or you'll get lost: Rational mutagenesis is a common approach to investigating or engineering enzyme function in vitro, but the ease with which one can manipulate protein sequences belies many pitfalls in connecting sparse activity data to an enzyme's true functional landscape. Using a high-throughput platform, Markin et al. expressed, purified, and performed an array of kinetic measurements on a target esterase, collecting data from >1000 mutations spanning the entire protein (see the Perspective by Baumer and Whitehead). Protein misfolding into an inactive state, rather than decreased equilibrium stability, was a crucial factor in negatively affected variants spread throughout the protein. When combined with prior mechanistic understanding and structures, four “functional components” help to rationalize the otherwise complex spatial pattern of effects of mutations on different aspects of enzyme function, all of which would be invisible from mutagenesis of just a few residues. Science , abf8761, this issue p. eabf8761 ; see also abj8346, p. 391

Short tandem repeats (STRs) are enriched in eukaryotic cis -regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)–DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis -regulatory mechanism to target TFs to genomic sites.

Fordyce et al. measure the sequence specificity and affinity of DNA-binding proteins using an improved microfluidic device. They characterize binding of 28 yeast transcription factors, several of which had proved intractable to previous approaches. Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide–major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR–T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3–specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.