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.

GMMA Can Stabilize Proteins Across Different Functional Constraints.

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.

Leaf Venation and Morphology Help Explain Physiological Variation in Yucca brevifolia and Hesperoyucca whipplei Across Microhabitats in the Mojave Desert, CA.

Different microclimates can have significant impact on the physiology of succulents that inhabit arid environments such as the Mojave Desert (California). We investigated variation in leaf physiology, morphology and anatomy of two dominant Mojave Desert monocots, Yucca brevifolia (Joshua tree) and Hesperoyucca whipplei, growing along a soil water availability gradient. Stomatal conductance (g s) and leaf thickness were recorded in the field at three different sites (north-western slope, south-eastern slope, and alluvial fan) in March of 2019. We sampled leaves from three individuals per site per species and measured in the lab relative water content at the time of g s measurements, saturated water content, cuticular conductance, leaf morphological traits (leaf area and length, leaf mass per area, % loss of thickness in the field and in dried leaves), and leaf venation. We found species varied in their g s: while Y. brevifolia showed significantly higher g s in the alluvial fan than in the slopes, H. whipplei was highest in the south-eastern slope. The differences in g s did not relate to differences in leaf water content, but rather to variation in number of veins per mm2 in H. whipplei and leaf width in Y. brevifolia. Our results indicate that H. whipplei displays a higher water conservation strategy than Y. brevifolia. We discuss these differences and trends with water availability in relation to species' plasticity in morphology and anatomy and the ecological consequences of differences in 3-dimensional venation architecture in these two species.