Essential role of the CD docking motif of MPK4 in plant immunity, growth, and development.

MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.

Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids.

Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.

Versatile control of the CDC48 segregase by the plant UBX-containing (PUX) proteins.

In plants, AAA-adenosine triphosphatase (ATPase) Cell Division Control Protein 48 (CDC48) uses the force generated through ATP hydrolysis to pull, extract, and unfold ubiquitylated or sumoylated proteins from the membrane, chromatin, or protein complexes. The resulting changes in protein or RNA content are an important means for plants to control protein homeostasis and thereby adapt to shifting environmental conditions. The activity and targeting of CDC48 are controlled by adaptor proteins, of which the plant ubiquitin regulatory X (UBX) domain-containing (PUX) proteins constitute the largest family. Emerging knowledge on the structure and function of PUX proteins highlights that these proteins are versatile factors for plant homeostasis and adaptation that might inspire biotechnological applications.

Synergy and allostery in ligand binding by HIV-1 Nef.

The Nef protein of human and simian immunodeficiency viruses boosts viral pathogenicity through its interactions with host cell proteins. By combining the polyvalency of its large unstructured regions with the binding selectivity and strength of its folded core domain, Nef can associate with many different host cell proteins, thereby disrupting their functions. For example, the combination of a linear proline-rich motif and hydrophobic core domain surface allows Nef to bind tightly and specifically to SH3 domains of Src family kinases. We investigated whether the interplay between Nef's flexible regions and its core domain could allosterically influence ligand selection. We found that the flexible regions can associate with the core domain in different ways, producing distinct conformational states that alter the way in which Nef selects for SH3 domains and exposes some of its binding motifs. The ensuing crosstalk between ligands might promote functionally coherent Nef-bound protein ensembles by synergizing certain subsets of ligands while excluding others. We also combined proteomic and bioinformatics analyses to identify human proteins that select SH3 domains in the same way as Nef. We found that only 3% of clones from a whole-human fetal library displayed Nef-like SH3 selectivity. However, in most cases, this selectivity appears to be achieved by a canonical linear interaction rather than by a Nef-like 'tertiary' interaction. Our analysis supports the contention that Nef's mode of hijacking SH3 domains is a virus-specific adaptation with no or very few cellular counterparts. Thus, the Nef tertiary binding surface is a promising virus-specific drug target.

Molecular basis for the adaptive evolution of environment-sensing by H-NS proteins.

The DNA-binding protein H-NS is a pleiotropic gene regulator in gram-negative bacteria. Through its capacity to sense temperature and other environmental factors, H-NS allows pathogens like Salmonella to adapt their gene expression to their presence inside or outside warm-blooded hosts. To investigate how this sensing mechanism may have evolved to fit different bacterial lifestyles, we compared H-NS orthologs from bacteria that infect humans, plants, and insects, and from bacteria that live on a deep-sea hypothermal vent. The combination of biophysical characterization, high-resolution proton-less nuclear magnetic resonance spectroscopy, and molecular simulations revealed, at an atomistic level, how the same general mechanism was adapted to specific habitats and lifestyles. In particular, we demonstrate how environment-sensing characteristics arise from specifically positioned intra- or intermolecular electrostatic interactions. Our integrative approach clarified the exact modus operandi for H-NS-mediated environmental sensing and suggested that this sensing mechanism resulted from the exaptation of an ancestral protein feature.

Methylation at the C-3' in D-Ring of Strigolactone Analogs Reduces Biological Activity in Root Parasitic Plants and Rice.

Strigolactones (SLs) regulate plant development and induce seed germination in obligate root parasitic weeds, e.g. Striga spp. Because organic synthesis of natural SLs is laborious, there is a large need for easy-to-synthesize and efficient analogs. Here, we investigated the effect of a structural modification of the D-ring, a conserved structural element in SLs. We synthesized and investigated the activity of two analogs, MP13 and MP26, which differ from previously published AR8 and AR36 only in the absence of methylation at C-3'. The de-methylated MP13 and MP26 were much more efficient in regulating plant development and inducing Striga seed germination, compared with AR8. Hydrolysis assays performed with purified Striga SL receptor and docking of AR8 and MP13 to the corresponding active site confirmed and explained the higher activity. Field trials performed in a naturally Striga-infested African farmer's field unraveled MP13 as a promising candidate for combating Striga by inducing germination in host's absence. Our findings demonstrate that methylation of the C-3' in D-ring in SL analogs has a negative impact on their activity and identify MP13 and, particularly, MP26 as potent SL analogs with simple structures, which can be employed to control Striga, a major threat to global food security.

H-NS uses an autoinhibitory conformational switch for environment-controlled gene silencing.

As an environment-dependent pleiotropic gene regulator in Gram-negative bacteria, the H-NS protein is crucial for adaptation and toxicity control of human pathogens such as Salmonella, Vibrio cholerae or enterohaemorrhagic Escherichia coli. Changes in temperature affect the capacity of H-NS to form multimers that condense DNA and restrict gene expression. However, the molecular mechanism through which H-NS senses temperature and other physiochemical parameters remains unclear and controversial. Combining structural, biophysical and computational analyses, we show that human body temperature promotes unfolding of the central dimerization domain, breaking up H-NS multimers. This unfolding event enables an autoinhibitory compact H-NS conformation that blocks DNA binding. Our integrative approach provides the molecular basis for H-NS-mediated environment-sensing and may open new avenues for the control of pathogenic multi-drug resistant bacteria.

Proteobacterial Origin of Protein Arginine Methylation and Regulation of Complex I Assembly by MidA.

The human protein arginine methyltransferase NDUFAF7 controls the assembly of the ∼1-MDa mitochondrial complex I (CI; the NADH ubiquinone oxidoreductase) by methylating its subunit NDUFS2. We determined crystal structures of MidA, the Dictyostelium ortholog of NDUFAF7. The MidA catalytic core domain resembles other eukaryotic methyltransferases. However, three large core loops assemble into a regulatory domain that is likely to control ligand selection. Binding of MidA to NDUFS2 is weakened by methylation, suggesting a mechanism for methylation-controlled substrate release. Structural and bioinformatic analyses support that MidA and NDUFAF7 and their role in CI assembly are conserved from bacteria to humans, implying that protein methylation already existed in proteobacteria. In vivo studies confirmed the critical role of the MidA methyltransferase activity for CI assembly, growth, and phototaxis of Dictyostelium. Collectively, our data elucidate the origin of protein arginine methylation and its use by MidA/NDUFAF7 to regulate CI assembly.