An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library.

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

Rationalizing Diverse Binding Mechanisms to the Same Protein Fold: Insights for Ligand Recognition and Biosensor Design.

The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem, which could enable many practical applications of protein biosensors. In this work, we analyzed two engineered biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands. MD simulations indicate that both PYR1 protein-ligand complexes bind a single conformer of their target ligand that is close to the lowest free-energy conformer. Computational design using a fixed conformer and rigid body orientation led to new WIN55,212-2 sensors with nanomolar limits of detection. This work reveals mechanisms by which the versatile PYR1 biosensor scaffold can bind diverse ligands. This work also provides computational methods to sample realistic ligand conformers and rigid body alignments that simplify the computational design of biosensors for novel ligands of interest.

Synthesis and characterization of abscisic acid receptor modulators.

The apocarotenoid phytohormone abscisic acid (ABA) regulates several aspects of plant development and stress responses. ABA is synthesized in response to multiple stressors and indirectly activates subfamily 2 Snf1-related kinases (SnRK2s) by receptor-mediated inhibition of clade A type IIC protein phosphatases (PP2Cs), which normally repress SnRK2 activity. The binding of ABA to its receptors triggers a change in receptor conformation that directs the formation of a receptor-ligand-PP2C complex that inhibits the activity of PP2C; this core mechanism can be harnessed for phosphatase activity-based measurements of receptor activation. In this chapter, we describe general methods for determining the effects of small molecules on ABA receptor function and supplement these with methods describing the synthesis of the high-affinity ligands opabactin (OP), which activates subfamily III and II ABA receptors, and the pan-receptor antagonist antabactin (ANT), and TAMRA-ANT fluorescent derivative of ANT. We present simple methods for quantifying receptor-ligand interactions using both PP2C inhibition and fluorescence polarization (FP) assays. Controls for determining the quality of recombinant receptors are provided, which are required for both quantitative analyses and for experimental consistency. Collectively, these methods will facilitate consistent biochemical measurements of the effects of ligand binding on ABA receptor function in vitro. Although the chapter describes ABA-specific methods, they illustrate and address a common need across receptor systems-reproducible assays that enable high throughput discovery and subsequent optimization of receptor modulators.

Dynamic control of plant water use using designed ABA receptor agonists.

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.

Functional analysis of synthetic DELLA domain peptides and bioactive gibberellin assay using surface plasmon resonance technology.

DELLA proteins and phytohormone gibberellin act together to control convergence point of plant development. A gibberellin-bound nuclear receptor that interacts with the N-terminal domain of DELLA proteins is required for gibberellin induced degradation of DELLA proteins. N-terminal DELLA domain includes two conserved motifs: DELLA and VHYNP. However, their respective functions remain unclear. Meanwhile, the identification and detection of several bioactive gibberellins from the more than 100 gibberellin metabolites are overwhelmingly difficult for their similar structures. Using in vitro biochemical approach, our work demonstrates for the first time that the synthetic GAI N-terminal DELLA domain peptides have similar bioactive function as the expressed protein to interact with AtGID1a receptor. Furthermore, our results reveal that DELLA motif is vitally important region and DELLA segment is essentially required region to recognize AtGID1a receptor. Finally, based on bioactive GA-dependent of the interaction between AtGID1a and DELLA protein, we generated a new method that could identify and detect bioactive GAs accurately and rapidly with surface plasmon resonance assays.

The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L.

Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1mM arsenate for 3weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography-a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an "artificial" database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata. BIOLOGICAL SIGNIFICANCE: Vacuole compartmentalization is crucial to As hyperaccumulator P. vittata, while there is limited known arsenic transport proteins involved in vacuole compartmentalization. In this paper, we obtained tonoplast of P. vittata fronds by iodixanol step gradient centrifugation method and then used TMPP protein labeling proteome technology for the quantitative detection of fronds tonoplast proteins. Our findings are the first challenge to the tonoplast proteins data mining of P. vittata which provide an informative basis for As carriers or channels. The proteomic approach in our study is suited for detecting alterations tonoplast protein and help to clarify the regulation of tonoplast transport processes. This article is part of a Special Issue entitled: Proteomics of non-model organisms.