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.     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -     

Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C‐terminal segment

The proliferating cell nuclear antigen (PCNA) is well recognized as one of the essential cellular components of the DNA replication machinery in all eukaryotic organisms. Despite their prominent importance, very little biochemical and structural information about plant PCNAs is available, in comparison with that obtained from other eukaryotic organisms. We have determined the atomic resolution crystal structures of the two distinct Arabidopsis thaliana PCNAs (AtPCNA), both complexed with the C‐terminal segment of human p21. Both AtPCNAs form homotrimeric ring structures, which are essentially identical to each other, including the major contacts with the p21 peptide. The structure of the amino‐terminal half of the p21 peptide, containing the typical PIP box sequence, is remarkably similar to those observed in the previously reported crystal structures of the human and archaeal PCNA‐PIP box complexes. Meanwhile, the carboxy‐terminal halves of the p21 peptide in the plant PCNA complexes are bound to the protein in a unique manner, most probably because of crystal packing effects. A surface plasmon resonance analysis revealed high affinity between each AtPCNA and the C‐terminal fragment of human p21. This result strongly suggests that the interaction is functionally significant, although no plant homologs of p21 have been identified yet. We also discovered that AtPCNA1 and AtPCNA2 form heterotrimers, implying that hetero‐PCNA rings may play critical roles in cellular signal transduction, particularly in DNA repair.     

Compact reduced thioredoxin structure from the thermophilic bacteria Thermus thermophilus

The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the "thioredoxin fold" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources.     

The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae

Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.     

Productive and nonproductive binding to ribonuclease A: X-ray structure of two complexes with uridylyl(2',5')guanosine

Guanine-containing mono- and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA. 1992. J Mol Biol 224:265-267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2',5')guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 A (R-factor 0.192) and 2.0 A (R-factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.     

Analysis of a new crystal form of procarboxypeptidase B: Further insights into the catalytic mechanism

A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen‐bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 178–185, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

Redox‐regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H‐site flexibility

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H₂O₂ concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox‐regulated enzyme.     

The structure of DesR from Streptomyces venezuelae,a β‐glucosidase involved in macrolide activation

Antibiotics have, indeed, altered the course of human history as is evidenced by the increase in human life expectancy since the 1940s. Many of these natural compounds are produced by bacteria that, by necessity, must have efficient self‐resistance mechanisms. The methymycin/pikromycin producing species Streptomyces venezuelae, for example, utilizes β‐glucosylation of its macrolide products to neutralize their effects within the confines of the cell. Once released into the environment, these compounds are activated by the removal of the glucose moiety. In S. venezuelae, the enzyme responsible for removal of the sugar from the parent compound is encoded by the desR gene and referred to as DesR. It is a secreted enzyme containing 828 amino acid residues, and it is known to be a retaining glycosidase. Here, we describe the structure of the DesR/D ‐glucose complex determined to 1.4‐Å resolution. The overall architecture of the enzyme can be envisioned in terms of three regions: a catalytic core and two auxiliary domains. The catalytic core harbors the binding platform for the glucose ligand. The first auxiliary domain adopts a “PA14 fold,” whereas the second auxiliary domain contains an immunoglobulin‐like fold. Asp 273 and Glu 578 are in the proper orientation to function as the catalytic base and proton donor, respectively, required for catalysis. The overall fold of the core region places DesR into the GH3 glycoside hydrolase family of enzymes. Comparison of the DesR structure with the β‐glucosidase from Kluyveromyces marxianus shows that their PA14 domains assume remarkably different orientations.     

The structure of a putative S‐formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C—O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.     

Synthesis, X-ray crystal structure and optical properties of novel 2-aryl-3-ethoxycarbonyl-4-phenylpyrido[1,2-a]benzimidazoles

A series of novel 2‐aryl‐3‐ethoxycarbonyl‐4‐phenylpyrido[1,2‐a]benzimidazole derivatives were synthesized by the tandem reaction of 2‐benzoyl benzimidazole and (Z)‐ethyl 4‐bromo‐3‐arylbut‐2‐enoate in the presence of potassium carbonate. The compounds were characterized using IR, ¹H‐NMR, ¹³C‐NMR, HRMS and the structure of 6f was further determined by X‐ray crystallography. Both absorption and fluorescence spectra characteristics of the compounds were investigated in acetonitrile and dichloromethane. The results showed that the absorption maxima of the compounds varied from 220 to 284 nm, depending on the structure of 2‐aryl group. The fluorescence results revealed that these compounds exhibited blue‐green fluorescence (463–475 nm) in dilute solutions and showed acceptable fluorescence quantum yields (Ф_PL = 0.13–0.73) in dichloromethane. Copyright © 2011 John Wiley & Sons, Ltd.     

X-ray analysis of phosphoglycerate kinase 2, a sperm-specific isoform from Mus musculus

Phosphoglycerate kinase 2 (PGK2) is an isozyme of the glycolytic pathway that provides ATP required for sperm motility. It is encoded by an autosomal retrogene that is expressed only during spermatogenesis, concomitant with the inactivation of the X-linked Pgk1 gene. PGK2 from the mouse, Mus musculus, has been overexpressed from a plasmid in bacteria and purified. It was crystallized in three forms: as the apoenzyme, as a complex with 3-phosphoglycerate (3PG), and as a complex with 3PG and ATP. The crystal structures were solved to 2.7, 2.0, and 2.7 A resolutions, respectively. The overall fold is nearly identical with previously solved mammalian PGK1 molecules. The apoenzyme is in the "open" form; that is the N-terminal domain that can bind 3PG and the C-terminal domain that binds ATP are too far apart for the substrates to interact. Binding 3PG causes a 13 degree rotation that partially closes the structure and causes helix 13, which is disordered in the unliganded structure, to stabilize. Binding ATP leaves the protein in the open configuration but also causes helix 13 to be ordered. Sequence alignment suggests that the active site of PGK2 is essentially identical to that of the cytoplasmic PGK1, but significant differences accumulate on a side of the C-terminal domain away from the active site. These changes may mediate the binding of this isoform to other proteins within the sperm flagellum, while still allowing the hinging action between the domains that is essential to catalytic activity.     

Conformational analysis of an acyclic tetrapeptide: ab‐initio structure determination from X‐ray powder diffraction,Hirshfeld surface analysis and electronic structure

A terminally protected acyclic tetrapeptide has been synthesized, and the crystal structure of its hydrated form, Boc‐Tyr‐Aib‐Tyr‐Ile‐OMe·2H₂O ( 1 ), has been determined directly from powder X‐ray diffraction data. The backbone conformation of tetrapeptide ( 1 ) exhibiting two consecutive β‐turns is stabilized by two 4 → 1 intramolecular N―H · · · O hydrogen bonds. In the crystalline state, the tetrapeptide molecules are assembled through water‐mediated O―H · · · O hydrogen bonds to form two‐dimensional molecular sheets, which are further linked by intermolecular C―H · · · O hydrogen bonds into a three‐dimensional supramolecular framework. The molecular electrostatic potential (MEP) surface of ( 1 ) has been used to supplement the crystallographic observations. The nature of intermolecular interactions in ( 1 ) has been analyzed quantitatively through the Hirshfeld surface and two‐dimensional fingerprint plot. The DFT optimized molecular geometry of ( 1 ) agrees closely with that obtained from the X‐ray structure analysis. The present structure analysis of Boc‐Tyr‐Aib‐Tyr‐Ile‐OMe·2H₂O ( 1 ) represents a case where ab‐initio crystal structure of an acyclic tetrapeptide with considerable molecular flexibility has been accomplished from laboratory X‐ray powder diffraction data. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structure of a purine/pyrimidine phosphoribosyltransferase-related protein from Thermus thermophilus HB8

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme involved in the salvage of adenine to form an adenine nucleotide. We crystallized and determined the X-ray crystallographic structure of a purine/pyrimidine phosphoribosyltransferase-related protein from the thermophilic bacterium, Thermus thermophilus HB8. The crystal space group was C2 with unit cell dimensions of a = 167.42 A, b = 61.41 A, c = 102.39 A, beta = 94.0 degrees . Initial phases were determined to 2.6 A using the multiple wavelength anomalous dispersion method and selenomethionine substituted protein (Se-MAD), and refined using a 1.9 A "native" data set. The asymmetric unit contains two pairs of identical dimers, each related by noncrystallographic two-fold symmetry. The fifth monomer forms a similar dimer across a crystallographic two-fold axis. These dimers appear to be the biological unit with both monomers contributing to an unusual highly charged arginine-rich bridge region separating the two active sites. Comparison with distantly related APRTases reveal similarities and differences of the active site.