41 research outputs found

    Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

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    The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

    Comparative Structural Analysis of Human DEAD-Box RNA Helicases

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    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members

    Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles

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    Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10–15 mM nitrate

    Crystallographic studies of enzymes involved in biosynthesis of fatty acids : Beta-ketoacyl acyl carrier protein synthase II and delta9-stearoyl-ACP desaturase

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    In the biosynthesis of fatty acids, the beta-Ketoacyl Acyl Carrier Protein Synthases (Kas) catalyse chain elongation by the addition of two-carbon units derived from malonyl-ACP, to an acyl group bound to CoA or the small protein ACP. These enzymes are potential targets for antibiotics against bacterial or parasitical infections and their mammalian counterparts could hopefully be used as drug targets against certain cancers and obesity. The crystal structure of the Kas II isoform from E. coli in complex with the well known natural inhibitor cerulenin has been determined to define the active site pocket and enable rational drug design with this target. Kas belongs to a family known as condensing enzymes for their ability to catalyse the Claisen condensation reaction. Different condensing enzymes have different substrate specificities and reaction mechanisms that somehow must be reflected in their 3D structure. The crystal structure of Kas II from Synechocystis was determined and compared to other condensing enzymes, to elucidate structural determinants for substrate specificity and reaction mechanism in condensing enzymes. Stearoyl Acyl Carrier Protein Desaturase is a soluble enzyme that catalyses the formation of a cis-double bond between carbon-9 and carbon-10 in stearic acid. Desaturase is known to contain a bent hydrophobic cavity, where the fatty acid substrate binds in the vicinity of a di-iron centre that is used for activation of molecular oxygen. Following reduction by ferredoxin, the di-iron centre activate molecular oxygen and reactive oxygen intermediates are formed that abstract hydrogen atoms from the bound fatty acid. The crystal structures of the molecular oxygen analogues, azide and acetate, in complex with desaturase have been determined in order to elucidate how the di-iron centre activates molecular oxygen and further controls the reactive oxygen intermediates to catalyse fatty acid desaturation. Crystal structures of iron depleted desaturase species have also been determined. Soluble desaturases act as the primary determinant for the composition of unsaturated fatty acids in plant membranes and seed storage oils, and variation in regio-specificity (double bond insertion position) among desaturase isozymes in certain plants is associated with the presence of unusual fatty acids in the seeds of these plants. The crystal structure of the protein complex between desaturase from Ricinus Communis and stearoyl-ACP from spinach, provides a basis for rational design of regio-specificity in soluble desaturases

    Optimization and application of the biotinylation approach for extraction and mass-spectrometric analysis of cell-surface associated proteins from Gram-positive bacteria

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    A method employing labeling of cell-surface proteins with Sulfo-NHS-SS-biotin and subsequent affinity enrichment with NeutrAvidin has been optimized in order to make cell-surface proteins from Gram-positive bacteria reliably accessible to quantitative mass spectrometric analyses. The optimized biotinylation approach was applied for analysis of the lipoproteome from S. aureus and S. pneumoniae on a global scale and the influence of mutations in the lipoprotein maturation pathway on the cell-surface and exoproteomes of both species was investigated. The biotinylation approach was integrated into a proteomic workflow that employs metabolic labeling with heavy nitrogen for relative protein quantification to investigate proteomic differences between S. aureus in a biofilm model and its free-floating, planktonic counterparts.Eine Methode wurde optimiert und angewendet, bei welcher zelloberflächenassoziierte Proteine mit Sulfo-NHS-SS-Biotin markiert werden, so dass sie mit NeutAvidin angereichert werden können, um sie für die quantitative massenspektrometrische Analyse zuverlässig zugänglich zu machen. Der optimierte Biotinylierungsansatz wurde dazu verwendet das Lipoproteom von S. aureus und S. pneumoniae global zu analysieren und den Einfluss von verschiedenen Mutationen im Reifungsprozess der Lipoproteine zu untersuchen. Der optimierte Biotinylierungsansatz wurde in eine Proteomanalyse integriert, die metabolisches Markieren der Proteine mit schwerem Stickstoff verwendet, um die Unterschiede im Proteom von S. aureus HG001 innerhalb von Biofilmen und freischwimmenden, planktonischen Zellen des gleichen Stammes herauszufinden

    Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51

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    Abstract FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer’s disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy

    Structures of BIR domains from human NAIP and cIAP2

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    The crystal structures of the human NAIP BIR2 and cIAP2 BIR3 domains have been determined. Both BIR domains harbors an amino-terminal tetrapeptide in its peptide-binding groove
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