Jill E. Gready

The C-type lectin-like domain superfamily

The superfamily of proteins containing C‐type lectin‐like domains (CTLDs) is a large group of extracellular Metazoan proteins with diverse functions. The CTLD structure has a characteristic double‐loop (‘loop‐in‐a‐loop’) stabilized by two highly conserved disulfide bridges located at the bases of the loops, as well as a set of conserved hydrophobic and polar interactions. The second loop, called the long loop region, is structurally and evolutionarily flexible, and is involved in Ca2+‐dependent carbohydrate binding and interaction with other ligands. This loop is completely absent in a subset of CTLDs, which we refer to as compact CTLDs; these include the Link/PTR domain and bacterial CTLDs. CTLD‐containing proteins (CTLDcps) were originally classified into seven groups based on their overall domain structure. Analyses of the superfamily representation in several completely sequenced genomes have added 10 new groups to the classification, and shown that it is applicable only to vertebrate CTLDcps; despite the abundance of CTLDcps in the invertebrate genomes studied, the domain architectures of these proteins do not match those of the vertebrate groups. Ca2+‐dependent carbohydrate binding is the most common CTLD function in vertebrates, and apparently the ancestral one, as suggested by the many humoral defense CTLDcps characterized in insects and other invertebrates. However, many CTLDs have evolved to specifically recognize protein, lipid and inorganic ligands, including the vertebrate clade‐specific snake venoms, and fish antifreeze and bird egg‐shell proteins. Recent studies highlight the functional versatility of this protein superfamily and the CTLD scaffold, and suggest further interesting discoveries have yet to be made.

Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas.

Picoeukaryotes are a taxonomically diverse group of organisms less than 2 micrometers in diameter. Photosynthetic marine picoeukaryotes in the genus Micromonas thrive in ecosystems ranging from tropical to polar and could serve as sentinel organisms for biogeochemical fluxes of modern oceans during climate change. These broadly distributed primary producers belong to an anciently diverged sister clade to land plants. Although Micromonas isolates have high 18S ribosomal RNA gene identity, we found that genomes from two isolates shared only 90% of their predicted genes. Their independent evolutionary paths were emphasized by distinct riboswitch arrangements as well as the discovery of intronic repeat elements in one isolate, and in metagenomic data, but not in other genomes. Divergence appears to have been facilitated by selection and acquisition processes that actively shape the repertoire of genes that are mutually exclusive between the two isolates differently than the core genes. Analyses of the Micromonas genomes offer valuable insights into ecological differentiation and the dynamic nature of early plant evolution.

Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein.

We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130-150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81-96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41-53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.

Comparative analysis of structural properties of the C-type-lectin-like domain (CTLD).

The superfamily of proteins containing the C‐type‐lectin‐like domain (CTLD) is a group of abundant extracellular metazoan proteins characterized by evolutionary flexibility and functional versatility. Several CTLDs are also found in parasitic prokaryotes and viruses. The 37 distinct currently available CTLD structures demonstrate significant structural conservation despite low or undetectable sequence similarity. Our aim in this study was to perform an extensive comparative analysis of all available CTLD structures to establish the most conserved structural features of the fold, and to test and extend the early analysis of Drickamer. By implication, these features should be those critical for maintenance of integrity of the fold. By analyzing CTLD structures superimposed by several methods, we have established groups of conserved structural positions involved in fold maintenance but not in ligand binding; these are consistent with the folds known functional flexibility. In addition to the well‐recognized disulfide bridges, groups of conserved residues are involved in hydrophobic interactions stabilizing the core of the fold and the long loop region, and in an α2‐β1–β5 polar interaction. Evaluation of the conclusions of the structure comparison study compared with alignments of all available human, mouse and Caenorhabditis elegans CTLD sequences showed that conservation patterns are preserved throughout the whole CTLD sequence space. Our observations provide an improved understanding of CTLD structure, and will help in identification of new CTLDs and the mechanisms that drive and constrain the coevolution of the structure and function of the fold. Proteins 2003;52:466–477.

Optimization of parameters for molecular dynamics simulation using smooth particle-mesh Ewald in GROMACS 4.5

Based on our critique of requirements for performing an efficient molecular dynamics simulation with the particle‐mesh Ewald (PME) implementation in GROMACS 4.5, we present a computational tool to enable the discovery of parameters that produce a given accuracy in the PME approximation of the full electrostatics. Calculations on two parallel computers with different processor and communication structures showed that a given accuracy can be attained over a range of parameter space, and that the attributes of the hardware and simulation system control which parameter sets are optimal. This information can be used to find the fastest available PME parameter sets that achieve a given accuracy. We hope that this tool will stimulate future work to assess the impact of the quality of the PME approximation on simulation outcomes, particularly with regard to the trade‐off between cost and scientific reliability in biomolecular applications.

Synthesis of quaternised 2-aminopyrimido[4,5-d]pyrimidin-4(3H)-ones and their biological activity with dihydrofolate reductase.

In a program to design and develop mechanism-based compounds active as substrates and inhibitors of dihydrofolate reductase (DHFR), we report the synthesis and physical properties of the 6-methyl- (7), 8-methyl- (8a), and 8-ethyl- (8b) derivatives of the parent 2-aminopyrimido[4,5-d]pyrimidin-4-(3H)-one (6). These compounds are the first members of a class of heterocycles related to 8-alkylpterins (N8-alkyl-2-aminopteridin-4(8H)-ones) (2a-2c), which have been shown to be novel substrates for DHFR. Three methods were developed for the synthesis of target compounds 7, 8a and 8b; however, the optimum yields (1-8%) could not be improved because the products decomposed by ring opening (e.g. to 2,4-diamino-5-methyliminomethylpyrimidin-6(1H)-one (9)) under the reaction conditions. The marked pi-electron deficiency of compounds 7, 8a and 8b is the likely cause for the susceptibility of the quaternised pyrimidine ring in the related cations 10, 15a and 15b, respectively, to add nucleophiles, thus promoting the opening of the pyrimidopyrimidine ring system. 1H-NMR spectroscopic studies of compounds 7, 8a and 8b revealed a fast and reversible covalent hydration of the associated cations across the C7z.sbnd;N8 bond for the N6-methyl derivative 7 and across the N6z.sbnd;C7 bond for the N8-methyl derivative 8a. UV spectroscopic studies of methyl derivatives 7 and 8a as well as the parent heterocycle 6 showed that protonation of the latter occurred at N1, while methylation with iodomethane proceeded at N6 and N8. The basicities of the N-methyl derivatives 7 and 8a (pK(a) ca. 5.5) are similar to those of 8-alkylpterins 2; this is an essential element of the design to promote binding to DHFR in their protonated form. Enzyme kinetics of 7, 8a and 8b with chicken DHFR confirmed our predictions that they are substrates, with apparent K(m) values of 3.8, 0.08, and 0.65 mM, and apparent V(max) values of 0.47, 2.27, and 0.30 nmol L(-1) min(-1) (for enzyme concentration 0.122 micro M), respectively. The parent compound 6 was not a substrate.

C-type lectin-like domains in Fugu rubripes

BackgroundMembers of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps).ResultsWe have performed an analysis of CTLD family composition in Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server http://anz.anu.edu.au:8080/Fugu_rubripes/ and their sequences as additional files with this paper.ConclusionsThe vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates.

Coupled semiempirical molecular orbital and molecular mechanics model (QM/MM) for organic molecules in aqueous solution

A coupled quantum mechanical and molecular mechanical (QM/MM) model based on the AM1, MNDO, and PM3 semiempirical molecular orbital methods and the TIP3P molecular mechanics model for liquid water is presented. The model was parameterized for each of the three molecular orbital methods using the aqueous solvation free energies of a wide range of neutral organic molecules, many of which are representative of amino acid side chains. The fit to the experimental solvation free energies was achieved by varying the radii in the van der Waals (vdW) terms for interactions between the solute, which was treated quantum mechanically, and the molecular mechanics (TIP3P) solvent molecules. It is assumed that the total free energy can be obtained as the sum of components derived from the electrostatic terms in the Hamiltonian plus a generally smaller “nonelectrostatic” term. The electrostatic contributions to the solvation free energies were computed using molecular dynamics (MD) simulation and thermodynamic integration techniques; the nonelectrostatic contributions were taken from the literature. It was found that the experimental free energies could be reproduced accurately (to within 1 kcal/mol) from the MD simulations, provided that the vdW parameter associated with hydrogen bonding (H bonding) was allowed to have different values depending on the QM method (AM1, MNDO, or PM3) and the type of functional group involved in the H bonding. Moreover, the radial distribution functions obtained from the MD simulations using such a parameterization scheme showed the expected H‐bonded structures between the solute and molecules of the solvent. The solvent‐induced dipole moments also compared favorably with the results of other QM/MM model calculations. © 1997 John Wiley & Sons, Inc. J Comput Chem 18: 1496–1512, 1997

Simple method for locating possible ligand binding sites on protein surfaces

A new, fast, and easy‐to‐implement method, van der Waals–fast Fourier transform (vdW‐FFT), for locating possible binding sites on the surface of a protein was developed and tested on a set of 15 different enzyme–ligand complexes. The method scans the whole protein surface and possible ligand orientations in order to find the best geometrical match, which corresponds to the minimum of the modified vdW energy. Two different grids, fine and coarse, and two sets of MM parameters, from the OPLS and Amber‐94 force fields, were used. The method has been shown to work accurately on the fine grid. On the coarse grid, the vdW‐FFT method failed only on two complexes. The C program implementing the method and test set of proteins is available free on our web site: http://biocomp.anu.edu.au/~aab20: 983–988, 1999

31P and 1H NMR spectroscopic studies of liver extracts of carbon tetrachloride-treated rats.

NMR spectroscopy was used to examine hepatic metabolism in cirrhosis with a particular focus on markers of functional cellular hypoxia. 31P and 1H NMR spectra were obtained from liver extracts from control rats and from rats with carbon tetrachloride‐induced cirrhosis. A decrease of 34% in total phosphorus content was observed in cirrhotic rats, parallelling a reduction of 40% in hepatocyte mass as determined by morphometric analysis. Hypoxia appeared to be present in cirrhotic rats, as evidenced by increased inorganic phosphate levels, decreased ATP levels, decreased ATP:ADP ratios (1.72 ± 0.40 vs 2.48 ± 0.50, p < 0.01), and increased inorganic phosphate:ATP ratios (2.77 ± 0.48 vs 1.62 ± 0.24, p < 0.00001). When expressed as a percentage of the total phosphorus content, higher levels of phosphoethanolamine and lower levels of NAD and glycerophosphoethanolamine were detected in cirrhotic rats. Cirrhotic rats also had increased phosphomonoester:phosphodiester ratios (5.73 ± 2.88 vs 2.53 ± 0.52, p < 0.01). These findings are indicative of extensive changes in cellular metabolism in the cirrhotic liver, with many findings attributable to the presence of intracellular hypoxia. Copyright