Implications for Chk1 Regulation: The 1.7 Å Crystal Structure of Human Cell Cycle Checkpoint Kinase Chk1

AbstractThe checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 Å resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity

Large-Scale Sequence Analysis of Hemagglutinin of Influenza A Virus Identifies Conserved Regions Suitable for Targeting an Anti-Viral Response

BACKGROUND: Influenza A viral surface protein, hemagglutinin, is the major target of neutralizing antibody response and hence a main constituent of all vaccine formulations. But due to its marked evolutionary variability, vaccines have to be reformulated so as to include the hemagglutinin protein from the emerging new viral strain. With the constant fear of a pandemic, there is critical need for the development of anti-viral strategies that can provide wider protection against any Influenza A pathogen. An anti-viral approach that is directed against the conserved regions of the hemaggutinin protein has a potential to protect against any current and new Influenza A virus and provide a solution to this ever-present threat to public health. METHODOLOGY/PRINCIPAL FINDINGS: Influenza A human hemagglutinin protein sequences available in the NCBI database, corresponding to H1, H2, H3 and H5 subtypes, were used to identify highly invariable regions of the protein. Nine such regions were identified and analyzed for structural properties like surface exposure, hydrophilicity and residue type to evaluate their suitability for targeting an anti-peptide antibody/anti-viral response. CONCLUSION/SIGNIFICANCE: This study has identified nine conserved regions in the hemagglutinin protein, five of which have the structural characteristics suitable for an anti-viral/anti-peptide response. This is a critical step in the design of efficient anti-peptide antibodies as novel anti-viral agents against any Influenza A pathogen. In addition, these anti-peptide antibodies will provide broadly cross-reactive immunological reagents and aid the rapid development of vaccines against new and emerging Influenza A strains

Genetic and Structure-Function Studies of Missense Mutations in Human Endothelial Lipase

<div><p>Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/β motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.</p> </div

Impact prediction of missense mutations on EL structure and function using CHARMM energies.

<p>Mutants for which lipase activity of EL were experimentally determined are shown in black (100% lipase activity: T111I, M361T), green (80–90% lipase activity: R54C, R389Q, N396S), yellow (55–60% lipase activity: G26S, E28K), and the predicted lipase activity for other mutations in red.</p

The back of EL structure contains a stretch of a sequence (312–340) with 10 Arginine (R) and 12 Lysine (K) and no negatively charged residues [Glutamic acid (E) or Aspartic acid (D)], which forms a positively charged groove, complementing negatively charged HSPG (A).

<p>Model of EL dimer with color-coded (purple) heparin binding sequence (<b>B</b>) and solvent accessible surface with docked decasaccharide (<b>C</b>). These structural studies predict how HSPG interacts with the EL homodimer.</p

Molecular model of EL and the location of all the known missense mutations (to date) on the structure.

<p>Molecular model of EL and the location of all the known missense mutations (to date) on the structure.</p

The effect of different EL-WT concentrations on HDL hydrolysis.

<p>The effect of different EL-WT concentrations on HDL hydrolysis.</p

HDL fatty acid composition after hydrolysis of HDL by EL in mutant (T338P) and in wild-type (A).

<p>Released HDL fatty acid by mutant EL (labeled red, n =  3) was compared to released HDL fatty acid by wild type EL (labeled green, n =  3) and to free fatty acids composition in untreated HDL (labeled blue, n =  3). The percentage decrease of fatty acids released from HDL by mutant EL compared to wild type (<b>B</b>). The fatty acids liberated by EL were quantified by subtraction of untreated HDL (negative control) data.</p

Multiple linear regression analyses of <i>LIPG</i> T111I SNP in the bi-ethnic population samples of San Luis Valley in Colorado.

<p>Multiple linear regression analyses of <i>LIPG</i> T111I SNP. The analyses were performed separately for Whites and Hispanics. The model is adjusted for gender, age, BMI, and smoking. The regression models were performed first for significant covariate(s) selection using stepwise variable selection. Then T111I SNP was added to the model that included significant covariates in an ‘enter’ method. Both genotypic and allelic association of the T111I with the lipid panel measurements were tested. For genotypic association, the genotypes were recoded in order to give weight to each genotype as following: CC = 0, CT = 1, TT = 2. For allelic association studies, dummy variables were created for the C/T alleles as following and both were included in the regression model: D_TT = 0 (for T/C and C/C genotypes); D_TT = 1 (for T/T genotype); D_TC = 0 (for T/T and C/C genotypes); D_TC = 1 (for T/C genotype). Similarly, dummy variables were created for covariate ‘smoking’ as following: D_Smoker = 0 (for nonsmokers and ex-smokers) and D_Smoker = 1 (for current smokers). B-value is the regression coefficient, which indicates the amount by which the tested variable is changed. The details of each significant linear regression model are provided under asterisk (*).</p