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.

Keywords: droplet microfluidics; histidine kinase; protein engineering; rational library design; signal transduction.

Fig. 1.

Generation of DNA libraries of predefined composition. Nonrandom walks through sequence space require libraries with predefined variants. (A) Visual representation of the sequence space. (A, Left) Combinations generated with a one-pot all-by-all approach yield, ideally, all combinations in the sequence space. (A, Right) Combinations generated by selectively combining specific parts allow predetermined walks through the sequence space and reduced screening effort. (B) Microfluidic approach for generating subset libraries. Switching rapidly between inlet channels combines parts and enzymes for assembly inside droplet reactors.

Fig. 2.

Hybrid microfluidic device that uses valves and droplets to deterministically combine and assemble DNA parts. (A) Schematic of the device showing fluidic and control layers, inlets, valves, and resistors. (B) Schematics and images of the fabricated device. The valve array contains 38 reagent valves leading to a common channel where reagents are combined and drops are formed. (C) The collection switch is a two-valve system for directing droplets to a collection tube or into waste. (D) Fluid flow rates in inlets with different locations on the main collection channel are equalized by pressure balancing with on-chip resistors.

Fig. 3.

Valve array duty cycle. (A) Valve opening and closing speed measured by fluorescence intensity. (B) Dye-labeled inlets show how reagents are combined in the central channel. Switching a single inlet from open (Top) to closed (Bottom) eliminates its stream from the central channel. (C) Flushing to waste reduces contamination from residual DNA parts from the prior cycle. (D) The device cycles between collecting drops (Top) and wasting drops (Bottom) as part of its duty cycle. Closed valves are colored black and open valves are colored white. Flowing DNA parts are colored blue, and blocked DNA parts are colored red. Enzymatic reagents are colored green. Oil is colored brown.

Fig. 4.

Construction and sequencing of the DNA library demonstrate efficient on-chip library subsetting. (A) Sequences of the four parts used to construct the combinatorial DNA library. (B, Top) Comparison of the synthetic and wild-type CpxA structures. (B, Bottom) The engineered CpxA contains MBP replacing the signal domain, followed by a two-helix transmembrane region encoded by parts A and B. The variable juxtamembrane linker (part C) between the TM and HAMP domain is followed by leucine substitutions in the S helix (part D). CpxA phosphorylates the response regulator CpxR, which activates transcription of a GFP reporter via the cpxP promoter. (C, Left) Rank-abundance curves comparing the full library generated on-chip (full), full library generated in a tube (tube), and subset library generated on-chip (subset). Microfluidic assembly enhances variant coverage compared with pooled, tube-based assembly. (C, Right) Subsampling reads and counting library members quantify diversity and confirm adequate sequencing depth. (D) Distribution of library sequences in the subset and full libraries, displayed as log2(read counts). Parts C and D are nested within parts A and B. (D, Left) AB combinations constrained by the length of A and B such that 18 AA ≤ [A + B] ≤ 25 AA (subset). (D, Right) No restriction is placed on which parts were combined to generate the library (full). (E) Sorting of GFP^high reporter cells expressing the CpxA library. Positive (CpxA L243S) and negative (CpxA Q229V) controls are shown.

Fig. 5.

Expression and screening of the DNA library encoding CpxA identify the S helix as a major determinant of signaling. (A) Enrichment as a function of leucine substitution (part D) for each alanine insertion (part C) (averaged over parts A and B). WT, wild type. (B) Enrichment as a function of linker alanine insertion (part C) for each Leu substitution (part D) (averaged over parts A and B). (C) Enrichment as a function of part A for each part B (averaged over parts C and D). (D) Enrichment as a function of part B for each part A (averaged over parts C and D). (A–D) Enrichment is calculated as the fold change in the normalized abundance of each variant sequence between post and presort. Error bars are SE, calculated as the mean divided by the square root of the sample size. Blue is the subset library and red is the full library. Q239L (full and subset) and 18 AA ≤ [A + B] ≤ 25 AA (subset) were removed from the library because of low counts. (E) Comparison of screen (enrichment) data with the functional cpxP::GFP assay. For the functional assay, L mutants are made in otherwise wild-type CpxA. (F) Location of the S helix in CpxA. (G) Mapping of reporter fluorescence (Top) and enrichment scores (Bottom) on the S helix of aligned CpxA structures (PDB ID codes 4BIV and 4BIU, chains A/B and D/E) shows enriched L variants segregate to the core of the dimer and deenriched variants segregate on the outward face.

Fig. 6.

Analysis of variable-length juxtamembrane Ala linkers (part C) in the context of different TM and S-helix domains. ABD sequences with similar variation in part C are clustered and enrichment is plotted as a function of the linker (part C). Sine curves fitted to data are superimposed on each dataset. The period of each sine curve falls within the expected range of 2.7 to 4.2, with few exceptions.

Fig. 7.

Structure–function relationships revealed by molecular dynamics simulations. (A) MD simulations predict all TM dimer geometries simulated in lipid bilayers, shown as cartoon ribbon main-chain traces (initial model, cyan; final MD frame, green; lipid headgroup phosphates, orange spheres). A sequence with the largest backbone rmsd of the final MD simulation frame (80 ns) versus the initial models is shown as a representative example. (B) Visual representation of the TM dimer and structural parameters extracted from MD simulation data. TM-domain structural features are predicted based on MD simulations for each AB combination (also see SI Appendix, Fig. S2F). (C) TM clustering based on structural features compared with TM clustering based on functional enrichment (screen). Functionally similar AB sequences segregate with structurally similar ones.