Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA

Abstract

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence–function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant “S helix” located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

“…BaeS is a closely related HK, which has significant overlap with CpxA, both in the inducing stimuli as well as the genes regulated ( Leblanc et al, 2011 ). We evaluated the activity of these kinases using previously validated fluorescent gene-reporters p cpxP ::GFP for CpxA activity ( Clark et al, 2021 ), p spy ::mCherry for BaeS activity Mensa et al, 2011 in a double CpxA/BaeS knockout strain.…”

Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code

“…CpxA was selected as the subject of QTY design, as it was one of the most studied SHKs 29 – 32 and explicitly confirmed to respond to the signals in vitro 33 . We applied the simple mode of QTY design to CpxA, i.e., substituting all transmembrane Leu (L), Ile (I), Val (V), and Phe (F) (Fig.…”

Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

“…BaeS is a closely related HK, which has significant overlap with CpxA, both in the inducing stimuli as well as the genes regulated (48). We evaluated the activity of these kinases using previously validated fluorescent gene-reporters (p cpxP ::GFP for CpxA activity(49), p spy ::mCherry for BaeS activity(50)) in a double CpxA/BaeS knockout strain. When the Gly 7 motif is inserted immediately upstream of the autokinase domain, we observe a high basal activity for both kinases similar to PhoQ ( Figure 5 ).…”