Thermostability of the PYL–PP2C Heterodimer Is Dependent on Magnesium: <i>In Silico</i> Insights into the Link between Heat Stress Response and Magnesium Deficiency in Plants

Magnesium deficiency increases the susceptibility of plants toward heat stress. The correlation between magnesium levels and stress response has been studied at the physiological level; albeit, the molecular explanation to this relationship remains elusive. Among diverse pathways implicated in the heat stress, the abscisic acid (ABA) signal modulates the heat stress response by magnesium dependent phosphatases (PP2Cs). Exclusively, sequestration of PP2Cs by ABA receptors (PYLs) in the heterodimer form activates the stress response through ABA responsive transcription factors. In this study, the molecular interplay between magnesium levels and ABA related heat stress response was investigated. Molecular dynamics simulations have been applied to two different PYL–PP2C heterodimer systems representing normal and magnesium deficient conditions. The heterodimer conformation and stability were delineated at high temperatures mimicking heat stress. Results showed that the thermostability of the heat stress response heterodimer was significantly dependent on the magnesium. Furthermore, a conserved aromatic cluster at the dimer interface acted synergistically with the metal to confer thermostability to the heterodimer structure. These structural insights into one of the possible links between magnesium levels and stress highlight the importance of metal micronutrients for tuning the stability of the stress-related proteins and optimizing tolerance

The structural impact of W211A mutation on the subunit interface.

<p>The subunit interface is rendered by van der Waals surface using the colors; green for chain A, blue for chain B, and <i>red</i> for 211 (W or A) that is found in chain B. The arrows indicate the residues that change conformation upon W211A mutation in (A) chain A and (B) chain B. The representation in (A) has been rotated 180° around the left diagonal axis to obtain (B), and the snapshots were taken from the simulations performed at 75°C.</p

Thermostability at different incubation concentrations.

<p>Residual lipase activity after thermal incubation for 30 minutes at concentrations of (A) 1 µM and (B) 50 µM. Student's t-test were performed to determine the significant differences in thermostability of W211A with respect to BTL2 (*p=0.1 and **p=0.05). </p

Zinc Modulates Self-Assembly of <i>Bacillus thermocatenulatus</i> Lipase

Thermoalkalophilic lipases are prone to aggregation from their dimer interface to which structural zinc is very closely located. Structural zinc sites have been shown to induce protein aggregation, but the interaction between zinc and aggregation tendency in thermoalkalophilic lipases remains elusive. Here we delineate the interplay between zinc and aggregation of the lipase from <i>Bacillus thermocatenulatus</i> (BTL2), which is taken to be a representative of thermoalkalophilic lipase. Results showed that zinc removal disrupted the BTL2 dimer, leading to monomer formation and reduced thermostability manifesting as a link between zinc and dimerization that leads to thermostability, while zinc addition induced aggregation. Biochemical and kinetic characterizations of zinc-induced aggregates showed that the aggregates obtained from the early and late stages of aggregation had differential characteristics. In the early stages, the aggregates were soluble and possessed native-like structures, while in the late stages, the aggregates became insoluble and showed fibrillar characteristics with binding affinities for Congo red and thioflavin T. The impact of temperature on zinc-induced aggregation was further investigated, and it was found that the native-like early aggregates could completely dissociate into functional lipase forms at high temperatures while dissociation of the late aggregates was limited. To this end, we report that the zinc-induced aggregation of BTL2 can be reversed by temperature switches and initiated by ordered aggregates in the early stages that gain fibrillar-like features over time. Insights revealed by this work contributes to the knowledge of aggregation mechanisms that exist in thermophilic proteins, reflecting the potential use of metal addition and/or removal to fine-tune aggregation tendency

The ANS fluorescence at 460 nm.

<p>The closed circles show the fluorescence from the lipases in 5 mM Tris-Cl at pH 7.0 and the open circles show the effect of 2-propanol. </p

The Conserved Lid Tryptophan, W211, Potentiates Thermostability and Thermoactivity in Bacterial Thermoalkalophilic Lipases

<div><p>We hypothesize that aggregation of thermoalkalophilic lipases could be a thermostability mechanism. The conserved tryptophans (W211, W234) in the lid are of particular interest owing to their previous involvements in aggregation and thermostability mechanisms in many other proteins. The thermoalkalophilic lipase from <i>Bacillus thermocatenulatus</i> (BTL2) and its mutants (W211A, W234A) were expressed and purified to homogeneity. We found that, when aggregated, BTL2 is more thermostable than its non-aggregating form, showing that aggregation potentiates thermostability in the thermoalkalophilic lipase. Among the two lid mutants, the W211A lowered aggregation tendency drastically and resulted in a much less thermostable variant of BTL2, which indicated that W211 stabilizes the intermolecular interactions in BTL2 aggregates. Further thermoactivity and CD spectroscopy analyses showed that W211A also led to a strong decrease in the optimal and the melting temperature of BTL2, implying stabilization by W211 also to the intramolecular interactions. The other lid mutant W234A had no effects on these properties. Finally, we analyzed the molecular basis of these experimental findings <i>in-silico</i> using the dimer (PDB ID: 1KU0) and the monomer (PDB ID: 2W22) lipase structures. The computational analyses confirmed that W211 stabilized the intermolecular interactions in the dimer lipase and it is critical to the stability of the monomer lipase. Explicitly W211 confers stability to the dimer and the monomer lipase through distinct aromatic interactions with Y273-Y282 and H87-P232 respectively. The insights revealed by this work shed light not only on the mechanism of thermostability and its relation to aggregation but also on the particular role of the conserved lid tryptophan in the thermoalkalophilic lipases.</p> </div

W211 in the active conformation.

<p>The domain formed by W211 in the open-active conformation (PDB ID: 2W22) is rendered by van der Waals surface. H87-G88 (green) and P232-V233-S236 (yellow) tightly packs the side chain of W211 colored in (red). </p

Crystal structure of the dimer BTL2.

<p>Two subunits, chain A and B are shown in black and silver, respectively, where the lids are colored in orange for both. The subunit interface is rendered by van der Waals surface: blue for chain A and <i>red</i> for chain B. The lid tryptophans W211 and W234 are shown in green sticks. (B) The aromatic cluster and (C) the network of hydrogen bonds were shown in stick models (C: cyan, N: blue, O: red).</p