Crystal structure of Arabidopsis thaliana casein kinase 2 α1.
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ABSTRACT:
SUBMITTER: Demulder M
PROVIDER: S-EPMC7137383 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory ?C helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.
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Project description:Casein kinase 1 (CK1) is an evolutionarily conserved protein kinase family among eukaryotes. Studies in non-plants have shown CK1-dependent divergent biological processes, but the collective knowledge regarding the biological roles of plant CK1 lags far behind other members of the Eukarya. One reason for this is that plants have many more genes encoding CK1 than do animals. To accelerate our understanding of the plant CK1 family, a strong CK1 inhibitor that efficiently inhibits multiple members of the CK1 protein family in vivo (i.e., in planta) is required. Here, we report a novel, specific, and effective CK1 inhibitor in Arabidopsis. Using circadian period-lengthening activity as an estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship study of analogues of the CK1 inhibitor PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride). A propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) had stronger CK1 inhibitory activity than PHA767491. A hybrid molecule of AMI-212 and AMI-23 (AMI-331) was about 100-fold more inhibitory than the parent molecule PHA767491. Affinity proteomics using an AMI-331 probe showed that the targets of AMI-331 inhibition are mostly CK1 kinases. As such, AMI-331 is a potent and selective CK1 inhibitor that shows promise in the research of CK1 in plants.
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Project description:The metabolism of sucrose is of crucial importance for life on Earth. In plants, enzymes called invertases split sucrose into glucose and fructose, contributing to the regulation of metabolic fluxes. Invertases differ in their localization and pH optimum. Acidic invertases present in plant cell walls and vacuoles belong to glycoside hydrolase family 32 (GH32) and have an all-? structure. In contrast, neutral invertases are located in the cytosol and organelles such as chloroplasts and mitochondria. These poorly understood enzymes are classified into a separate GH100 family. Recent crystal structures of the closely related neutral invertases InvA and InvB from the cyanobacterium Anabaena revealed a predominantly ?-helical fold with unique features compared with other sucrose-metabolizing enzymes. Here, a neutral invertase (AtNIN2) from the model plant Arabidopsis thaliana was heterologously expressed, purified and crystallized. As a result, the first neutral invertase structure from a higher plant has been obtained at 3.4?Å resolution. The hexameric AtNIN2 structure is highly similar to that of InvA, pointing to high evolutionary conservation of neutral invertases.
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Project description:Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-L-homoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 A resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.
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Project description:Transmembrane kinases (TMKs) are members of the plant receptor-like kinase (RLK) family. TMKs are characterized by an extracellular leucine-rich-repeat (LRR) domain, a single transmembrane region and a cytoplasmic kinase domain. TMKs have been shown to act as critical modulators of cell expansion and cell proliferation. Here, the crystal structure of the extracellular domain of TMK3 (TMK3-ECD) was determined to a resolution of 2.06 Å, with an Rwork of 17.69% and an Rfree of 20.58%. Similar to the extracellular domain of TMK1, the TMK3-ECD structure contains two solenoids with 13 LRRs and a non-LRR region (316-364) between the tenth and 11th LRRs. A comparison of TMK3-ECD with other LRR-RLKs that contain a non-LRR region indicates that the non-LRR region plays a critical role in structural integrity and may contribute to ligand interactions. The non-LRR region of TMK3-ECD is characterized by two disulfide bonds that may have critical biological implications.
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Project description:Peroxiredoxins (Prxs), a large family of antioxidant enzymes, are abundant in all living organisms. Peroxiredoxin A (PrxA) from Arabidopsis thaliana belongs to the typical 2-Cys Prx family and is localized in the chloroplast. This article reports the crystal structure of a PrxA C119S mutant refined to 2.6?Å resolution. The protein exists as a decamer both in the crystal structure and in solution. The structure is in the reduced state suitable for the approach of peroxide, though conformational changes are needed for the resolving process.
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Project description:The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 Å resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an ?-helical-rich C-terminus and characteristic N-terminal left-handed parallel ?-helix (L?H). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the L?H not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.
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Project description:Glutamate-1-semialdehyde-2,1-aminomutase (GSAM) catalyzes the isomerization of glutamate-1-semialdehyde (GSA) to 5-aminolevulinate (ALA) and is distributed in archaea, most bacteria and plants. Although structures of GSAM from archaea and bacteria have been resolved, a GSAM structure from a higher plant is not available, preventing further structure-function analysis. Here, the structure of GSAM from Arabidopsis thaliana (AtGSA1) obtained by X-ray crystallography is reported at 1.25?Å resolution. AtGSA1 forms an asymmetric dimer and displays asymmetry in cofactor binding as well as in the gating-loop orientation, which is consistent with previously reported Synechococcus GSAM structures. While one monomer binds PMP with the gating loop fixed in the open state, the other monomer binds either PMP or PLP and the gating loop is ready to close. The data also reveal the mobility of residues Gly163, Ser164 and Gly165, which are important for reorientation of the gating loop. Furthermore, the asymmetry of the AtGSA1 structure supports the previously proposed negative cooperativity between monomers of GSAM.
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Project description:We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.
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