A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

Phenotypic and metabolic plasticity shapes life-history strategies under combinations of abiotic stresses.

Plants developed various reversible and non-reversible acclimation mechanisms to cope with the multifaceted nature of abiotic-stress combinations. We hypothesized that in order to endure these stress combinations, plants elicit distinctive acclimation strategies through specific trade-offs between reproduction and defense. To investigate Brachypodium distachyon acclimation strategies to combinations of salinity, drought and heat, we applied a system biology approach, integrating physiological, metabolic, and transcriptional analyses. We analyzed the trade-offs among functional and performance traits, and their effects on plant fitness. A combination of drought and heat resulted in escape strategy, while under a combination of salinity and heat, plants exhibited an avoidance strategy. On the other hand, under combinations of salinity and drought, with or without heat stress, plant fitness (i.e., germination rate of subsequent generation) was severely impaired. These results indicate that under combined stresses, plants' life-history strategies were shaped by the limits of phenotypic and metabolic plasticity and the trade-offs between traits, thereby giving raise to distinct acclimations. Our findings provide a mechanistic understanding of plant acclimations to combinations of abiotic stresses and shed light on the different life-history strategies that can contribute to grass fitness and possibly to their dispersion under changing environments.

Tighter αC-helix-αL16-helix interactions seem to make p38α less prone to activation by autophosphorylation than Hog1.

Many eukaryotic protein kinases (EPKs) are autoactivated through autophosphorylation of their activation loop. Mitogen-activated protein (MAP) kinases do not autophosphorylate spontaneously; relying instead upon mitogen-activated protein kinase (MAPK) kinases (MKKs) for their activation loop phosphorylation. Yet, in previous studies we identified mutations in the yeast MAPK high osmolarity glycerol (Hog1) that render it capable of spontaneous autophosphorylation and consequently intrinsically active (MKK-independent). Four of the mutations occurred in hydrophobic residues, residing in the αC-helix, which is conserved in all EPKs, and in the αL16-helix that is unique to MAPKs. These four residues interact together forming a structural element termed 'hydrophobic core'. A similar element exists in the Hog1's mammalian orthologues p38s. Here we show that the 'hydrophobic core' is a loose suppressor of Hog1's autophosphorylation. We inserted 18 point mutations into this core, 17 of which were able to render Hog1 MKK-independent. In p38s, however, only a very few mutations in the equivalent residues rendered these proteins intrinsically active. Structural analysis revealed that a salt bridge between the αC-helix and the αL16-helix that exists in p38α may not exist in Hog1. This bond further stabilizes the 'hydrophobic core' of p38, making p38 less prone to de-repressing its concealed autophosphorylation. Mutating equivalent hydrophobic residues in Jnk1 and Erk2 has no effect on their autophosphorylation. We propose that specific structural elements developed in the course of evolution to suppress spontaneous autophosphorylation of Hog1/p38. The suppressors were kept wobbly, probably to allow activation by induced autophosphorylation, but became stricter in mammalian p38s than in the yeast Hog1.

Chimeric avidin shows stability against harsh chemical conditions--biochemical analysis and 3D structure.

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.

Critical importance of loop conformation to avidin-enhanced hydrolysis of an active biotin ester.

The homotetrameric and biotin-binding properties of avidin and streptavidin have been exploited for a myriad of biotechnological applications and theoretical studies. Among the few differences between the two proteins is the capacity of avidin to hydrolyze biotinyl p-nitrophenyl ester (BNP), as opposed to streptavidin, which fully protects the same pseudosubstrate from hydrolysis. Combined mutagenesis and X-ray analysis have been used to attempt to understand this diametric difference in activities. It was found that a charged residue and one of the loops (L3,4) are together responsible for this difference. Recently, the avidin-related analogue AVR4 was found to have an even more pronounced BNP-hydrolysis activity than avidin. Again, the combination of charged residue(s) (Asp39 and/or Arg112) and the rigid conformation of the L3,4 loop was suggested to be responsible for the observed hydrolysis reaction. However, replacement of the latter charged residues in AVR4 resulted in only a modest reduction in hydrolytic activity at most, whereas replacement of the L3,4 loop of avidin with the rigid loop of AVR4 caused a dramatic increase in the activity of avidin. These results clearly demonstrate that the main feature responsible for the observed differences in rates of hydrolysis among the avidins is the conformational status of the L3,4 loop, which imposes conformational constraints on the pseudosubstrate, thereby rendering it susceptible to nucleophilic attack by solvent. In this context, the hydrolytic properties of the avidins reflect enzyme catalysis, in that subtleties in substrate binding are the determining features of catalytic efficiency.