Plant immunophilins: a review of their structure-function relationship.

BACKGROUND: Originally discovered as receptors for immunosuppressive drugs, immunophilins consist of two major groups, FK506 binding proteins (FKBPs) and cyclosporin A binding proteins (cyclophilins, CYPs). Many members in both FKBP and CYP families are peptidyl prolyl isomerases that are involved in protein folding processes, though they share little sequence homology. It is not surprising to find immunophilins in all organisms examined so far, including viruses, bacteria, fungi, plants and animals, as protein folding represents a common process in all living systems. SCOPE OF REVIEW: Studies on plant immunophilins have revealed new functions beyond protein folding and new structural properties beyond that of typical PPIases. This review focuses on the structural and functional diversity of plant FKBPs and CYPs. MAJOR CONCLUSIONS: The differences in sequence, structure as well as subcellular localization, have added on to the diversity of this family of molecular chaperones. In particular, the large number of immunophilins present in the thylakoid lumen of the photosynthetic organelle, promises to deliver insights into the regulation of photosynthesis, a unique feature of plant systems. However, very little structural information and functional data are available for plant immunophilins. GENERAL SIGNIFICANCE: Studies on the structure and function of plant immunophilins are important in understanding their role in plant biology. By reviewing the structural and functional properties of some immunophilins that represent the emerging area of research in plant biology, we hope to increase the interest of researchers in pursuing further research in this area. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis.

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.

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.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38).

AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83-437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 A resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C222(1), with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 A, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 A at the NSLS synchrotron. The structure is being solved by the MAD method.

Structural and pharmacological comparison of daboiatoxin from Daboia russelli siamensis with viperotoxin F and vipoxin from other vipers.

Russell's viper (Vipera russelli, also known as Daboia russelli) is one of the major causes of fatal snakebites. To date, five Daboia russelli subspecies have been recognized. Daboiatoxin (DbTx) is the main lethal phospholipase A(2) (PLA(2)) toxin in the venom of D. russelli siamensis (Myanmar viper) and has strong neurotoxic, myotoxic and cytotoxic activities. DbTx and its homologous neurotoxins viperotoxin F from D. russelli formosensis (Taiwan viper) and vipoxin from the Bulgarian sand viper V. ammodytes meridionalis consist of complexes between a nontoxic acidic PLA(2) protein and an enzymatically active basic PLA(2). DbTx and viperotoxin F are presynaptic toxins, while vipoxin is postsynaptic. The two chains of DbTx have been separated and their PLA(2) enzymatic activity has been measured using the secretory PLA(2) assay kit. The enzymatic activity of DbTx chain B is reduced by 30% of its original activity by chain A in a unimolar ratio, thus indicating that DbTx chain A acts as an inhibitor. The lethal activity of the two chains has also been studied in male albino mice and chain A is less lethal than chain B. The crystal structure of DbTx has also been determined and its structural details are compared with those of the two homologues. Furthermore, an attempt is made to correlate the sequence and structural determinants of these toxins with their enzymatic activities and their pharmacological effects.

Structural analysis uncovers a role for redox in regulating FKBP13, an immunophilin of the chloroplast thylakoid lumen.

Change in redox status has long been known to link light to the posttranslational regulation of chloroplast enzymes. So far, studies have been conducted primarily with thioredoxin-linked members of the stroma that function in a broad array of biosynthetic and degradatory processes. Consequently, little is known about the role of redox in regulating the growing number of enzymes found to occur in the lumen, the site of oxygen evolution in thylakoid membranes. To help fill this gap, we have studied AtFKBP13, an FKBP-type immunophilin earlier shown to interact with a redox-active protein of the lumen, and found the enzyme to contain a pair of disulfide bonds in x-ray structural studies. These disulfides, which in protein mutagenesis experiments were shown to be essential for the associated peptidyl-prolyl isomerase activity, are unique to chloroplast FKBPs and are absent in animal and yeast counterparts. Both disulfide bonds were redox-active and were reduced by thioredoxin from either chloroplast or bacterial sources in a reaction that led to loss of enzyme activity. The results suggest a previously unrecognized paradigm for redox regulation in chloroplasts in which activation by light is achieved in concert with oxygen evolution by the oxidation of sulfhydryl groups (conversion of SH to S-S). Such a mechanism, occurring in the thylakoid lumen, is in direct contrast to regulation of enzymes in the stroma, where reduction of disulfides targeted by thioredoxin (S-S converted to SH) leads to an increase in activity in the light.