The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue α-mannan backbone repeating unit, consisting of a triad of α-(1→3)-mannoses, modified by ß-(1→2)-xyloses on the first two mannoses and a ß-(1→2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Glycan Stability and Flexibility: Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens.

We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.

The Incorporation of Labile Protons into Multidimensional NMR Analyses: Glycan Structures Revisited.

Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longer-lived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H2O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans' labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss "active" glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2-8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY's NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 µs molecular dynamics simulations.

Characterization of the ß-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide.

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gram-negative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identified as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1 → 5)-α-KDOp-(2 → partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.

Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn-to-Ala substitutions at the predicted N-glycosylation sites of the M41-RBD were evaluated along with two control Val-to-Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41-RBD construct. Six of the 10 glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented α2,3-linked sialic acid oligosaccharide ligand. LC/MSE glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall, the glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41-RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein.

The ß-reducing end in α(2-8)-polysialic acid constitutes a unique structural motif.

Over the years, structural characterizations of α(2-8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is ß, which has the [I]CO2- group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2- group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2-8)-polySia near the reducing end, as the ß configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2-). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-ßOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2-8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2-8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.

Glycan OH Exchange Rate Determination in Aqueous Solution: Seeking Evidence for Transient Hydrogen Bonds.

Hydrogen bonds (Hbonds) are important stabilizing forces in biomolecules. However, for glycans in aqueous solution, direct NMR detection of Hbonds is elusive because of their transient nature. Here, we present Isotope-based Natural-abundance TOtal correlation eXchange SpectroscopY (INTOXSY), a new 1H-13C heteronuclear single quantum coherence-total correlation spectroscopy based method, to extract OH groups' exchange rate constants (kex) for molecules in natural 13C abundance and show that OH Hbonds can be inferred from "slower" H/D kex. We evaluate kex measured with INTOXSY in light of those extracted with line-shape analysis. Subsequently, we use a set of common glycans to establish a kex reference basis set and to infer the existence of transient Hbonds involving OH donor groups. Then, we report kex values for a series of mono- and disaccharides, as well as for oligosaccharides sialyl Lewis X and ß-cyclodextrin, and compare the results with those from the reference set to extract Hbond information. Finally, we utilize NMR experimental data in conjunction with molecular dynamics simulations to establish donor and acceptor Hbond pairs. Our exchange rate measurements indicate that OH/OD exchange rates, kHD, values <10 s-1 are consistent with transient Hbond OH groups and potential acceptor groups can be uncovered through MD simulations.

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.

NMR of glycans: shedding new light on old problems.

The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.

Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates.

Carbon plays a central role in the molecular architecture of carbohydrates, yet the availability of accurate methods for (1)D(CC) determination has not been sufficiently explored, despite the importance that such data could play in structural studies of oligo- and polysaccharides. Existing methods require fitting intensity ratios of cross- to diagonal-peaks as a function of the constant-time (CT) in CT-COSY experiments, while other methods utilize measurement of peak separation. The former strategies suffer from complications due to peak overlap, primarily in regions close to the diagonal, while the latter strategies are negatively impacted by the common occurrence of strong coupling in sugars, which requires a reliable assessment of their influence in the context of RDC determination. We detail a (13)C-(13)C CT-COSY method that combines a variation in the CT processed with diagonal filtering to yield (1)J(CC) and RDCs. The strategy, which relies solely on cross-peak intensity modulation, is inspired in the cross-peak nulling method used for J(HH) determinations, but adapted and extended to applications where, like in sugars, large one-bond (13)C-(13)C couplings coexist with relatively small long-range couplings. Because diagonal peaks are not utilized, overlap problems are greatly alleviated. Thus, one-bond couplings can be determined from different cross-peaks as either active or passive coupling. This results in increased accuracy when more than one determination is available, and in more opportunities to measure a specific coupling in the presence of severe overlap. In addition, we evaluate the influence of strong couplings on the determination of RDCs by computer simulations. We show that individual scalar couplings are notably affected by the presence of strong couplings but, at least for the simple cases studied, the obtained RDC values for use in structural calculations were not, because the errors introduced by strong couplings for the isotropic and oriented phases are very similar and therefore cancel when calculating the difference to determine (1)D(CC) values.

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.

Nanoscale fullerene compression of an yttrium carbide cluster.

The nanoscale parameters of metal clusters and lattices have a crucial influence on the macroscopic properties of materials. Herein, we provide a detailed study on the size and shape of isolated yttrium carbide clusters in different fullerene cages. A family of diyttrium endohedral metallofullerenes with the general formula of Y(2)C(2n) (n = 40-59) are reported. The high field (13)C nuclear magnetic resonance (NMR) and density functional theory (DFT) methods are employed to examine this yttrium carbide cluster in certain family members, Y(2)C(2)@D(5)(450)-C(100), Y(2)C(2)@D(3)(85)-C(92), Y(2)C(2)@C(84), Y(2)C(2)@C(3v)(8)-C(82), and Y(2)C(2)@C(s)(6)-C(82). The results of this study suggest that decreasing the size of a fullerene cage with the same (Y(2)C(2))(4+) cluster results in nanoscale fullerene compression (NFC) from a nearly linear stretched geometry to a constrained "butterfly" structure. The (13)C NMR chemical shift and scalar (1)J(YC) coupling parameters provide a very sensitive measure of this NFC effect for the (Y(2)C(2))(4+) cluster. The crystal structural parameters of a previously reported metal carbide, Y(2)C(3) are directly compared to the (Y(2)C(2))(4+) cluster in the current metallofullerene study.

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.

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.

Backbone (1)H, (15)N, and (13)C 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 (1)H, (15)N, and (13)C 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.

89Y and 13C NMR cluster and carbon cage studies of an yttrium metallofullerene family, Y3N@C(2n) (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.

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).

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.