Hugo F. Azurmendi

89Y and 13C NMR Cluster and Carbon Cage Studies of an Yttrium Metallofullerene Family, Y3N@C2n (n = 40−43)

The members of a new family of yttrium trimetallic nitride-templated (TNT) endohedral metallofullerenes (EMFs), Y(3)N@C(2n) (n = 40-43), have been synthesized and purified. On the basis of experimental and computational (13)C NMR studies, we propose cage structures for Y(3)N@I(h)-C(80) (IPR allowed), Y(3)N@D(5h)-C(80) (IPR allowed), Y(3)N@C(s)-C(82) (non-IPR), Y(3)N@C(s)-C(84) (non-IPR), and Y(3)N@D(3)-C(86) (IPR allowed). A significant result is the limited number of isomers found for each carbon cage. For example, there are 24 isolated pentagon rule (IPR) and 51 568 non-IPR structures possible for the C(84) cage, but only one major isomer of Y(3)N@C(s)-C(84) was found. The current study confirms the unique role of the trimetallic nitride (M(3)N)(6+) cluster template in the Kratschmer-Huffman electric-arc process for fullerene cage size and high symmetry isomer selectivity. This study reports the first (89)Y NMR results for Y(3)N@I(h)-C(80,) Y(3)N@C(s)(51365)-C(84), and Y(3)N@D(3)(19)-C(86), which reveal a progression from isotropic to restricted (Y(3)N)(6+) cluster motional processes. Even more surprising is the sensitivity of the (89)Y NMR chemical shift parameter to subtle changes in the electronic environment at each yttrium nuclide in the (Y(3)N)(6+) cluster (more than 200 ppm for these EMFs). This (89)Y NMR study suggests that (89)Y NMR will evolve as a powerful tool for cluster motional studies of EMFs.

Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.

Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.

Pseudocryptand-Type [2]Pseudorotaxanes Based on Bis(meta-phenylene)-32-Crown-10 Derivatives and Paraquats with Remarkably Improved Association Constants

The first dual component pseudocryptand-type [2]pseudorotaxanes were designed and prepared via the self-assembly of synthetically easily accessible bis(meta-phenylene)-32-crown-10 pyridyl, quinolyl, and naphthyridyl derivatives with paraquat. The formation of the pseudocryptand structures in the complexes remarkably improved the association constant by forming the third pseudobridge via H-bonding with the guest and π-stacking of the heterocyclic units.

Syntheses and Structures of Phenyl-C81-Butyric Acid Methyl Esters (PCBMs) from M3N@C80

Two new 6,6-open phenyl-C(81)-butyric acid methyl ester metallofulleroids, M(3)N@C(80)PCBM (M = Sc, Y), were synthesized by diazoalkane addition reactions and fully characterized. The results demonstrate that the reactive sites are the same for M(3)N@C(80) (M = Sc, Y) but dramatically different from that of C(60).

The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides

TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.

Structural and membrane binding properties of the prickle PET domain.

The planar cell polarity (PCP) pathway is required for fetal tissue morphogenesis as well as for maintenance of adult tissues in animals as diverse as fruit flies and mice. One of the key members of this pathway is Prickle (Pk), a protein that regulates cell movement through its association with the Dishevelled (Dsh) protein. Pk presents three LIM domains and a PET domain of unknown structure and function. Both the PET and LIM domains control membrane targeting of Dsh, which is necessary for Dsh function in the PCP pathway. Here, we show that the PET domain is monomeric and presents a nonglobular conformation with some properties of intrinsically disordered proteins. The PET domain adopts a helical conformation in the presence of 2,2,2-trifluoroethanol (TFE), a solvent known to stabilize hydrogen bonds within the polypeptide backbone, as analyzed by circular dichroism (CD) and NMR spectroscopy. Furthermore, we found that the conserved and single tryptophan residue in PET, Trp 536, moves to a more hydrophobic environment when accompanied with membrane penetration and that the protein becomes more helical in the presence of lipid micelles. The presence of LIM domains, downstream of PET, increases protein folding, thermostability, and tolerance to limited proteolysis. In addition, pull-down and tryptophan fluorescence analyses suggest that the LIM domains physically interact to regulate membrane penetration of the PET domain. The findings reported here favor a model where the PET domain is engaged in Pk membrane insertion, whereas the LIM domains modulate this function.

Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl Lewis-X.

We describe the direct NMR detection of a C-H···O nonconventional hydrogen bond (Hbond) and provide experimental and theoretical evidence for conventional Hbonds in the pentasaccharide sialyl Lewis-X (sLe(X)-5) between 5 and 37 °C in water. Extensive NMR structural studies together with molecular dynamics simulations offer strong evidence for significant local dynamics in the Le(X) core and for previously undetected conventional Hbonds in rapid equilibrium that modulate structure. These NMR studies also showed temperature-dependent (1)H and (13)C line broadening. The resulting model emerging from this study is more complex than a simple rigid core description of Le(X)-like molecules and improves our understanding of stabilizing interactions in glycans.

Backbone 1H, 15N, and 13C resonance assignments and secondary structure of the tollip CUE domain

The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1-binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone 1H, 15N, and 13C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response.

CHAPTER 1:Intramolecular Hydrogen Bonding in Glycans in Aqueous Solution

Hydrogen bonding is a vital feature of biomolecular structure. Hydrogen bonds help proteins, DNA and RNA fold, giving rise to their shape and are thus an important factor in molecular recognition. Hydrogen bonds have been identified in aqueous solutions in proteins and nucleic acids, however, they have not been detected in aqueous solutions of glycans. In this chapter, we discuss the detection of hydrogen bonds in aqueous solution by NMR spectroscopy. These include NH-, OH- and CH-based hydrogen bonds. We describe methods for their detection and the types of hydrogen bonds that have been identified in glycans thus far. We also show how hydrogen bonds in glycans help form helices and other structures, which may affect the shape of these glycans and thus contribute to their flexibility and function.