Trazel Teh

Mammalian AMP-activated Protein Kinase Subfamily

The mammalian 5′-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (α1) is distinct from the previously cloned kinase subunit (α2). The α1 (548 residues) and α2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase α1 mRNA distribution and the α1 immunoreactivity rather than α2, with substantial amounts in kidney, liver, lung, heart, and brain. Both α1 and α2 isoforms are stimulated by AMP and contain noncatalytic β and γ subunits. The liver α1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the α2 immunoreactivity parallels the α2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the β and γ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Turn up the HEAT.

The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead‐associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130–140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55–75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non‐catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH4). The crystal structure of PAH revealed that the N‐terminal sequence of the enzyme (residues 19–29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N‐terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH4. Our data support the model where the N‐terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.

Crystallization of importin α, the nuclear-import receptor

Crystals of recombinant importin alpha, the nuclear-import receptor, have been obtained at two different pH conditions by vapour diffusion using sodium citrate as precipitant and dithiothreitol as an additive. At pH 4-5, the crystals have the symmetry of the trigonal space group P3121 or P3221 (a = b = 78.0, c = 255.8 A, gamma = 120 degrees ); at pH 6-7, the crystals have the symmetry of the orthorhombic space group P212121 (a = 78.5, b = 89.7, c = 100.5 A). In both cases, there is probably one molecule of importin alpha in the asymmetric unit. At least one of the crystal forms diffracts to a resolution higher than 3 A using the laboratory X-ray source; the crystals are suitable for crystal structure determination.

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP‐dependent protein kinase at Ser16 in the N‐terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N‐terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N‐terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N‐terminal region, but no sharp resonances are present in a truncation mutant lacking the N‐terminal 29 residues. The N‐terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2–20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N‐terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

The use of Co2+ for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein.

Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 A) is compatible with the Cu K alpha wavelength (1.54 A) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein-protein interaction domains of approximately 130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 127.3, c = 386.3 A; diffraction data have been collected to 3.1 A resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 A.