Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The DeltaPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y(D)(ox) radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S(2)Q(A)(-) and S(2)Q(B)(-) charge recombinations were stabilized in DeltaPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y(D)(+)Q(A)(-) recombination, pointed to the donor side modifications in DeltaPsbR. EPR measurements revealed that S(1)-to-S(2)-transition and S(2)-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q(A) to Q(B) electron transfer in DeltaPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

Biochemical Studies Provide Insights into the Necessity for Multiple Arabidopsis thaliana Protein-Only RNase P Isoenzymes

RNase P catalyzes removal of the 5′ leader from precursor tRNAs (pre-tRNAs) in all three domains of life. Some eukaryotic cells contain multiple forms of the protein-only RNase P (PRORP) variant, prompting efforts to unravel this seeming redundancy. Previous studies concluded that there were only modest differences in the processing of typical pre-tRNAs by the three isoforms in Arabidopsis thaliana [AtPRORP1 (organellar), AtPRORP2 and AtPRORP3 (nuclear)]. Here, we investigated if different physical attributes of the three isoforms might engender payoffs under specific conditions. Our temperature–activity profiling studies revealed that AtPRORPs display substrate-identity dependent behavior at elevated temperatures (37–45 °C), with the organellar variant outperforming the nuclear counterparts. Echoing these findings, molecular dynamics simulations revealed that AtPRORP2 relative to AtPRORP1 samples a wider conformational ensemble that deviates from the crystal structure. Results from our biochemical studies and molecular dynamics simulations support the idea that AtPRORPs have overlapping but not necessarily redundant attributes and inspire new perspectives on the suitability of each variant to perform its function(s) in a specific cellular locale.     

Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ～19.80 mm, V(max) ～0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (～40% for AtClpC2 and ～20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.     

Insights into the functional properties of the marneral oxidase CYP71A16 from Arabidopsis thaliana

The Arabidopsis thaliana gene encoding CYP71A16 is part of the gene cluster for the biosynthesis and modification of the triterpenoid marneral. Previous investigations of A. thaliana have revealed that CYP71A16 catalyzes marneral oxidation, while it also can accept marnerol as substrate. The aim of the present study was to investigate functional properties of CYP71A16 in vitro. For this purpose, heterologous expression of a N-terminally modified version of CYP71A16 was established in Escherichia coli, which yielded up to 50 mg L^− 1 recombinant enzyme. The enzyme was purified and activity was reconstituted in vitro with different redox partners. A heterologous bacterial redox partner system consisting of the flavodoxin YkuN from Bacillus subtilis and the flavodoxin reductase Fpr from E. coli clearly outperformed the cytochrome P450 reductase ATR2 from A. thaliana in supporting the CYP71A16-mediated hydroxylation of marnerol. Substrate binding experiments with CYP71A16 revealed a dissociation constant K_D of 225 μM for marnerol. CYP71A16 catalyzed the hydroxylation of marnerol to 23-hydroxymarnerol with a K_M of 142 μM and a k_cat of 3.9 min^− 1. Furthermore, GC/MS analysis revealed an as of yet unidentified overoxidation product of this in vitro reaction. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.     

Structural and Functional Studies of the Mitochondrial Cysteine Desulfurase from Arabidopsis thaliana

AtNfs1 is the Arabidopsis thaliana mitochondrial homolog of the bacterial cysteine desulfurases NifS and IscS, having an essential role in cellular Fe-S cluster assembly. Homology modeling of AtNfs1m predicts a high global similarity with E. coli IscS showing a full conservation of residues involved in the catalytic site, whereas the chloroplastic AtNfs2 is more similar to the Synechocystis sp. SufS. Pull-down assays showed that the recombinant mature form, AtNfs1m, specifically binds to Arabidopsis frataxin (AtFH). A hysteretic behavior, with a lag phase of several minutes, was observed and hysteretic parameters were affected by pre-incubation with AtFH. Moreover, AtFH modulates AtNfs1m kinetics, increasing V (max) and decreasing the S (0.5) value for cysteine. Results suggest that AtFH plays an important role in the early steps of Fe-S cluster formation by regulating AtNfs1 activity in plant mitochondria.     

Structural comparison of oxidized and reduced FKBP13 from Arabidopsis thaliana

AtFKBP13, an immunophilin in the chloroplast thylakoid lumen, participates in redox-regulatory processes via a pair of conserved disulfide bonds that are present at the N- and C-termini of the protein. Characterization of this protein by structural and biochemical analysis has revealed a novel mechanism of redox regulation in the thylakoid lumen. The protein is active in its oxidized form but is inactivated after reduction by the thioredoxin system. This is in sharp contrast with the regulation of biosynthetic enzymes in the stroma of the chloroplast, where reduction of enzymes by thioredoxin activates their function. To understand how the reduced form of AtFKBP13 is stabilized and how reduction of the cysteine residues affects the molecular properties of the enzyme, we determined the crystal structure of reduced AtFKBP13 at 1.88 A. Comparison of the reduced structure and the oxidized form that we published earlier shows rearrangements in redox site regions, readjustments of hydrogen-bonding interactions and the secondary structure of the active site residues 50-53, and reduced accessibility of the catalytic residues involved in the peptidyl proline isomerase (PPIase) activity of this enzyme. We propose that redox-linked changes in the secondary structure of the PPIase domain are responsible for significant functional differences in this protein in the reduced and oxidized states.     

Transfer of the maize transposable element MU1 into Arabidopsis thaliana

The maize transposable element Mu1 was transferred to Arabidopsis thaliana by Ti plasmid-mediated transformation and a fertile line containing Mu1 was regenerated. Southern analysis of transformed tissue indicated that the Mu1 DNA remained entirely within a segment of T-DNA during three sexual generations. The results of a search for spontaneous mutations in a large number of Mu1-containing seedlings suggests that the presence of Mu1 did not cause a major increase in the spontaneous mutation frequency.     

Structural Insights into the Mechanisms and Pharmacology of K2P Potassium Channels

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K_2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K_2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K_2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K_2P function, uncovered the importance of asymmetric pinching and dilation motions in the K_2P selectivity filter (SF) C-type gate, and defined two K_2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K_2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K_2P activity. Such knowledge should catalyze development of new K_2P modulators to probe function and treat a wide range of disorders.     

Peroxidase activity of annexin 1 from Arabidopsis thaliana

On the basis of earlier reports suggesting that annexin A1 from Arabidopsis thaliana (AnnAt1) participates in limiting the excessive levels of reactive oxygen species during oxidative burst in plants, we examined the sensitivity of recombinant AnnAt1 to hydrogen peroxide and its peroxidase activity. Purified recombinant protein remains mostly alpha-helical and binds to lipids in a calcium-dependent manner. Upon oxidation recombinant AnnAt1 exhibits a tendency to form dimers in vitro. AnnAt1 is also sensitive to the presence of reducing agents, suggesting that AnnAt1 is a redox sensor in plant cells. Moreover, using two independent methods we found that AnnAt1 displayed peroxidase activity which is probably related to the presence of a heme-binding domain within AnnAt1, as present in other peroxidases. Indeed, site-directed mutagenesis within this domain resulted in a complete abrogation of the activity of AnnAt1. Furthermore, this activity was found to be sensitive to the phosphorylation state of the protein.     

Structural Insights into the Assembly of CARMA1 and BCL10

The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 Å. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.