Kristallstrukturanalyse des kohlenhydratbindenden Moduls 27-1 der Beta-Mannanase 26 aus Caldicellulosiruptor saccharolyticus im Komplex mit Mannohexaose und Kristallisation der ATPase HP0525 aus Helicobacter pylori

Kohlenhydrat-bindende Module (CBMs) sind die bekanntesten nicht-katalytischen Module, die mit Enzymen assoziiert sind, welche die pflanzliche Zellwand hydrolysieren. Die beta-Mannanase 26 von Caldicellulosiruptor saccharolyticus, Stamm Rt8B.4, ist eine thermostabile modulare Glycosidhydrolase, die N-terminal zwei dicht aufeinander folgende nicht-katalytische kohlenhydratbindende Module besitzt. Diese spezifisch beta-Mannan bindenden CBMs wurden kürzlich als Mitglieder der CBM-Familie 27 klassifiziert. Im ersten Teil dieser Arbeit wird die Kristallisation und Strukturanalyse des ersten kohlenhydratbindenden Moduls der ß-Mannanase aus C. saccharolyticus (CsCBM27-1) mit einer gebundenen Mannohexaose und in ligandfreier Form beschrieben. Grundlage für diese Arbeit waren Daten aus der isothermen Titrationskalorimetrie zur Quantifizierung der Affinität von CsCBM27-1 für lösliche Mannooligosaccharide. Die hier präsentierte hochaufgelöste Kristallstruktur des ungebundenen und Mannohexaose gebundenen CsCBM27-1 erlaubt weitere Einblicke in die Interaktion ß-Mannan bindender CBMs mit ihren entsprechenden Liganden. CsCBM27-1 zeigt eine typische ß-sandwich jellyroll-Struktur mit gebundenen Kalziumion. Die Mannohexaosebindung wird durch drei dem Lösungsmittel zugängliche Tryptophanreste und einige direkte Wasserstoffbrückenbindungen vermittelt. Der zweite Teil der Arbeit beschäftigt sich mit der Reinigung und Kristallisation der ATPase Virb11 HP0525 aus Helicobacter pylori. Das native Protein HP0525 ließ sich gut rekombinant herstellen und reinigen. Es wurde aus einer von mehreren Kristallisationsbedingungen durch Optimierung der Kristallisationskomponenten ausreichend große Kristalle erhalten, die gute Diffraktionseigenschaften zeigten. Neben dem nativen Protein wurde Selenomethionin-substituiertes Protein synthetisiert und gereinigt. Von diesem Protein SeMet-HP0525, resultierten hexagonale Kristalle. Zur Derivat-Datensatzsammlung ist es aufgrund der Publikation der Kristallstruktur dieser hexameren ATPase HP0525 nicht mehr gekommen. Weitere strukturelle Untersuchungen an diesem Protein wurden als nicht mehr erforderlich angesehen.Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Caldicellulosiruptor saccharolyticus strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase beta-mannanase which contains two non-catalytic modules in tandem at its N-terminus. These modules were recently shown to function primarily as ß-mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. In the first part of this study, the crystallization and crystal structure analysis of the first carbohydrate binding module (CsCBM27-1) of the beta-mannanase from C. saccharolyticus in native and mannohexaose-bound form is described. The basis for this study were data from isothermal titration calorimetry for quantifying the binding affinity of CsCBM27-1 for soluble mannooligosaccharidesBoth structures permit further insights into the interaction of beta-mannan binding CBMs with their corresponding ligands. CsCBM27-1 shows the typical beta-sandwich jellyroll fold observed in other CBMs with a single calcium ion bound opposite to the ligand binding site. This arrangement is similar to topologies of other CBM families. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. The second part of this study addressed the purification and crystallization of the VirB11 ATPase HP0525 of Helicobacter pylori. The native HP0525 protein was produced in recombinant Escherichia coli and purified for crystallization. One of several crystallization experiments yielded large crystals by optimization of the concentration of the crystallization components. The crystals revealed good diffraction behavior. In addition to the native protein, selenomethionine-substituted HP0525 was produced and purified. Hexagonal crystals were obtained from the SeMet-HP0525. No derivative datasets were collected, because the crystal structure of the hexameric ATPase HP0525 was published by Yeo et al. (2000). Further structural investigations for the protein HP0525 were judged unnecessary

Unusual Armadillo Fold in the Human General Vesicular Transport Factor p115

The golgin family gives identity and structure to the Golgi apparatus and is part of a complex protein network at the Golgi membrane. The golgin p115 is targeted by the GTPase Rab1a, contains a large globular head region and a long region of coiled-coil which forms an extended rod-like structure. p115 serves as vesicle tethering factor and plays an important role at different steps of vesicular transport. Here we present the 2.2 Å-resolution X-ray structure of the globular head region of p115. The structure exhibits an armadillo fold that is decorated by elongated loops and carries a C-terminal non-canonical repeat. This terminal repeat folds into the armadillo superhelical groove and allows homodimeric association with important implications for p115 mediated multiple protein interactions and tethering

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5′ exonuclease-mediated nucleic acid degradation

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3

PIN and CCCH Zn-finger domains coordinate RNA targeting in ZC3H12 family endoribonucleases.

The CCCH-type zinc finger (ZnF) containing ZC3H12 ribonucleases are crucial in post-transcriptional immune homoeostasis with ZC3H12A being the only structurally studied member of the family. In this study, we present a structural-biochemical characterization of ZC3H12C, which is linked with chronic immune disorders like psoriasis. We established that the RNA substrate is cooperatively recognized by the PIN and ZnF domains of ZC3H12C and analyzed the crystal structure of ZC3H12C bound to a single-stranded RNA substrate. The RNA engages in hydrogen-bonded contacts and stacking interactions with the PIN and ZnF domains simultaneously. The ZC3H12 ZnF shows unprecedented structural features not previously observed in any member of the CCCH-ZnF family and utilizes stacking interactions via a unique combination of spatially conserved aromatic residues to align the target transcript in a bent conformation onto the ZnF scaffold. Further comparative structural analysis of ZC3H12 CCCH-ZnF suggests that a trinucleotide sequence is recognized by ZC3H12 ZnF in target RNA. Our work not only describes the initial structure-biochemical study on ZC3H12C, but also provides the first molecular insight into RNA recognition by a ZC3H12 family member. Finally, our work points to an evolutionary code for RNA recognition adopted by CCCH-type ZnF proteins

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

BACKGROUND: Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs). RESULTS: Good quality crystals that were suitable for X-ray crystallographic studies were obtained following the truncation of the 14 N-terminal amino acids of APEG-1, a region predicted to be disordered. The truncated protein (termed ΔAPEG-1) consists of a single immunoglobulin (Ig) like domain which includes an Arg-Gly-Asp (RGD) adhesion recognition motif. The RGD motif is crucial for the interaction of extracellular proteins and plays a role in cell adhesion. The X-ray structure of ΔAPEG-1 was determined and was refined to sub-atomic resolution (0.96 Å). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key β-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the β-sheets form a hydrophobic core. The RGD motif folds into a 3(10 )helix that is involved in the formation of a homodimer in the crystal which is mainly stabilized by salt bridges. Analytical ultracentrifugation studies revealed a moderate dissociation constant of 20 μM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength. CONCLUSION: Our data suggests that the RGD motif might play a role not only in the adhesion of extracellular proteins but also in intracellular protein-protein interactions. However, it remains to be established whether the rather weak dimerisation of APEG-1 involving this motif is physiogically relevant

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer

TapA acts as specific chaperone in TasA filament formation by strand complementation

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell–cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface

TapA acts as specific chaperone in TasA filament formation by strand complementation

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell–cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface

Structural basis of phosphatidylinositol 3-kinase C2α function

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide–MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease