Structural basis of autoregulation of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Active site-directed protein regulation

Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effecters bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

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 FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Formal methods within a totally functional approach to programming

Taking functional programming to its extremities in search of simplicity still requires integration with other development (e.g. formal) methods. Induction is the key to deriving and verifying functional programs, but can be simplified through packaging proofs with functions, particularly folds, on data (structures). Totally Functional Programming avoids the complexities of interpretation by directly representing data (structures) as platonic combinators - the functions characteristic to the data. The link between the two simplifications is that platonic combinators are a kind of partially-applied fold, which means that platonic combinators inherit fold-theoretic properties, but with some apparent simplifications due to the platonic combinator representation. However, despite observable behaviour within functional programming that suggests that TFP is widely-applicable, significant work remains before TFP as such could be widely adopted.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Population structure of Phytophthora cinnamomi in South Africa

Phytophthora cinnamomi isolates collected from 1977 to 1986 and 1991 to 1993 in two regions in South Africa were analyzed using isozymes. A total of 135 isolates was analyzed for 14 enzymes representing 20 putative loci, of which four were polymorphic. This led to the identification of nine different multilocus isozyme genotypes. Both mating types of P. cinnamomi occurred commonly in the Cape region, whereas, predominantly, the A2 mating type occurred in the Mpumalanga region of South Africa. A2 mating type isolates could be resolved into seven multilocus isozyme genotypes, compared with only two multilocus isozyme genotypes for the A1 mating type isolates. Low levels of gene (0.115) and genotypic (2.4%) diversity and a low number of alleles per locus (1.43) were observed for the South African P. cinnamomi population. The genetic distance between the Cape and Mpumalanga P. cinnamomi populations was relatively low (D-m = 0.165), and no specific pattern in regional distribution of multilocus isozyme genotypes could be observed. The genetic distance between the ''old'' (isolated between 1977 and 1986) and ''new'' (isolated between 1991 and 1993) P. cinnamomi populations from the Cape was low (D-m = 0.164), indicating a stable population over time. Three of the nine multilocus isozyme genotypes were specific to the ''old'' population, and only one multilocus isozyme genotype was specific to the ''new'' population. Significant differences in allele frequencies, a high genetic distance (D-m = 0.581) between the Cape A1 and A2 mating type isolates, significant deviations from Hardy-Weinberg equilibrium, a low overall level of heterozygosity, and a high fixation index (0.71) all indicate that sexual reproduction occurs rarely, if at all, in the South African P. cinnamomi population.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as `benign cytochrome c oxidase deficiency myopathy` is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T > C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Influence of Biliary Drainage on the Repair of Hepatic Lesions in Biliary Fibrosis

Background. Bilioduodenal (BD) and biliojejunal (BJ) derivation induce enterobiliary reflux and bile stasis. Decompression of the excluded loop of the Roux-en-Y (BJD) was proposed to minimize these effects. The aim of this study was to compare the influence of these three modalities of biliary bypass on hepatic lesion repair in rats with secondary biliary fibrosis. Materials and Methods. Rats with 15 d of biliary obstruction underwent BD, BJ, and BJD drainage and were compared with a group submitted to simulated operation (SO) and biliary obstruction (CBO). The serum values of total and fractional bilirubin, alkaline phosphatase (ALP), and aminotransferases (AST and ALT), as well as hepatobiliointestinal excretion determined with (99m)Tc-Disida, were used for comparison. In addition, we used morphometric analyses to estimate the mass of the hepatocytes, bile ducts, and liver fibrosis. We also counted hepatic stellate cells (SC). Results. For each of the three modalities of biliary drainage, there were significant reductions in bilirubin, AST, ALP, and the number of SCs. The recovery of the estimated mass of all histologic components occurred only after BJ and BJD; in the BD group, the estimated hepatocyte mass was reduced compared with the SO group. The residual hepatic radioactivity of (99m)Tc-Disida was greater in the BJD group than in the SO group. Conclusions. The interposition of the jejunal loop between the biliary tree and the intestine may slow hepatobiliary clearance of radioactivity, even though it provides the resolution of cholestasis and is effective in recovering from hepatic lesions. (C) 2011 Elsevier Inc. All rights reserved.