Homonuclear NOESY Research Articles

A concise route to fused tetrazolo scaffolds through 10-camphor sulfonic acid auto-tandem homogeneous catalysis and mechanistic investigation

10-Camphor sulfonic acid (10-CSA) as an organo-catalyst has gained interest due to its versatile solubility and easiness of handling. This work reports a simple synthetic method through non-classical Biginelli for the construction of tetrazolo pyrimidine (4a-m) and quinazolines (4a′-o′). Azolopyrimidines and quinazolines are of great pharmaceutical importance. Numerous compounds are currently in use for the treatment of different diseases. Therefore their synthesis is industrially inevitable. Employing aldehydes, 1,3-dicarbonyls, and 5-Aminotetrazole, we report eco-friendly, cost-effective catalysis through a tandem reaction catalyzed by the 10-CSA that gave excellent yields, 70–95 % for tetrazolo quinazoline and 45–76 % tetrazolo pyrimidines respectively. The homonuclear NOESY analysis confirms the selective formation of one isomer. All the compounds are characterised by 1H NMR, 13C NMR, and MS. Investigation of the reaction mechanism by both experimental and theoretical studies provides evidence. Mechanism of the reaction was also explained utilizing the information from mass spectrometry monitoring. DFT calculation carried out at PBEPBE (Perdew-Burke-Ernzerhof) functional and 6-31G (d,p) basis set level of theory of the various intermediates observed supports the experimental evidence.

SAT-040 Structural Mechanisms of Parathyroid Hormone (PTH) Binding to Receptor and Regulation of Endosomal Signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR primarily activates the stimulatory G-protein, Gs, for adenylate cyclases that induce the production of cAMP. Our laboratory has demonstrated that PTH stimulates both transient and prolonged cAMP production at the plasma membrane and in endosomes, respectively. Activation of PKA through PTHR signaling in endosomes activates v-ATPases, which acidify the endosomal lumen. A decrease below pH 6.5 triggers the release of PTH from the receptor, thereby terminating Gs signaling. However, the structural mechanisms of PTH binding to PTHR and release from endosomal receptor are unclear. We studied the structural changes of PTH(1-34) upon binding to PTHR using nuclear magnetic resonance (NMR). We acquired 1H-15N HSQC, 2D 1H homonuclear NOESY, TOCSY, and 3D 15N-edited NOESY NMR spectra. The Hα chemical shifts of 15N-labeled PTH(1-34) residues, compared to “random coil” chemical shifts, demonstrate that at plasma membrane pH, unbound PTH consists of two halves: a helical C-terminal half (residues 18-34) and a less structured N-terminal half (1-13) connected by a random coil “hinge” (14-17). We hypothesize this hinge coordinates a two-step ligand binding mechanism, in which the helical C-terminal half of PTH binds to the extracellular domain of PTHR (ECD), followed by binding of the N-terminal half to the receptor transmembrane domain (TMD) and subsequent receptor activation. To investigate the first step of ligand binding, we compared the 1H-15N TROSY spectra of 15N-PTH(1-34) in the absence and presence of unlabeled ECD. HSQC peak intensities of PTH residues 21-34 are significantly reduced in the presence of ECD, suggesting that the flexibility of helical PTH(21-34) is compromised upon binding to ECD. Interestingly, peaks of L15 and S17, which participate in the hinge, disappear in the presence of ECD. In addition, new peaks for several PTH residues in both N- and C-terminal halves appear in the presence of ECD, indicating that ECD binding triggers new conformations of these residues. These data suggest that the increased rigidity of the hinge and new conformations of other N-terminal half residues position the N-terminal half for interaction with and binding to the TMD. To investigate the structural changes of PTH during endosomal acidification, we acquired HSQC spectra at pH 7.2, 6.5, and 5.9. These data reveal that the chemical shifts of PTH residues H9, H14, L15, and H32 are significantly affected by pH decrease, with H14 and L15 notably being most affected. Since H14 and L15 participate the hinge, we predict that conformational changes in the hinge as a result of pH decrease trigger ligand dissociation. Our work gives insight into the structural mechanisms of PTHR ligand binding and signaling regulation.

Structural mechanisms of parathyroid hormone (PTH) binding to receptor and regulation of endosomal signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR primarily activates the stimulatory G‐protein, Gs, for adenylate cyclases that induces the production of cAMP. Our laboratory has demonstrated that PTH stimulates both transient and prolonged cAMP production at the plasma membrane and in endosomes, respectively. Activation of PKA through PTHR signaling in endosomes activates v‐ATPases, which acidify the endosomal lumen. A decrease below pH 6.5 triggers the release of PTH from the receptor, thereby terminating Gs signaling. However, the structural mechanisms of PTH binding to PTHR and release from endosomal receptor are unclear. We studied the structural changes of PTH(1‐34) upon binding to PTHR using nuclear magnetic resonance (NMR). We acquired 1H‐15N HSQC, 2D 1H homonuclear NOESY, TOCSY, and 3D 15N‐edited NOESY NMR spectra. The H〈 chemical shifts of 15N‐labeled PTH(1‐34) residues, compared to “random coil” chemical shifts, demonstrate that at plasma membrane pH, unbound PTH consists of two halves: a helical C‐terminal half (residues 18–34) and a less structured N‐terminal half (1‐13) connected by a random coil “hinge” (14–17). We hypothesize this hinge coordinates a two‐step ligand binding mechanism, in which the helical C‐terminal half of PTH binds to the extracellular domain of PTHR (ECD), followed by binding of the N‐terminal half to the receptor transmembrane domain (TMD) and subsequent receptor activation. To investigate the first step of ligand binding, we compared the 1H‐15N TROSY spectra of 15N‐PTH(1‐34) in the absence and presence of unlabeled ECD. HSQC peak intensities of PTH residues 21‐34 are significantly reduced in the presence of ECD, suggesting that the flexibility of helical PTH(21‐34) is compromised upon binding to ECD. Interestingly, peaks of L15 and S17, which participate in the hinge, disappear in the presence of ECD. In addition, new peaks for several PTH residues in both N‐ and C‐terminal halves appear in the presence of ECD, indicating that ECD binding triggers new conformations of these residues. These data suggest that the increased rigidity of the hinge and new conformations of other N‐terminal half residues position the N‐terminal half for interaction with and binding to the TMD. To investigate the structural changes of PTH during endosomal acidification, we acquired HSQC spectra at pH 7.2, 6.5, and 5.9. These data reveal that the chemical shifts of PTH residues H9, H14, L15, and H32 are significantly affected by pH decrease, with H14 and L15 notably being most affected. Since H14 and L15 participate in the hinge, we predict that conformational changes in the hinge as a result of pH decrease trigger ligand dissociation. Our work gives insight into the structural mechanisms of PTHR ligand binding and signaling regulation.Support or Funding InformationNIH R01 DK116780 and DK102495This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemical Synthesis of Glycoproteins with the Specific Installation of Gradient‐Enriched 15N‐Labeled Amino Acids for Getting Insights into Glycoprotein Behavior

Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial 15 N-labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen 15 N-labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of 15 N atoms. This idea discriminated even the same type of amino acid based on the intensities of 1 H-15 N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied circular dichroism (CD) spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor.

Detection of intermolecular transferred NOEs in large protein complexes using asymmetric deuteration: HIV-1 gp120 in complex with a CCR5 peptide.

Weak protein-protein and protein-ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27-residue peptide (deuterated at Ile and Val residues) corresponding to the N-terminal segment of the human C-C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4-mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope-edited/isotope-filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off-rates.

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear 1H NOESY spectra, with chemical shifts, 3JHNHα scalar couplings, residual dipolar couplings, and 1H-15N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.

Norditerpenoid Alkaloids from Delphinium flexuosum Bieb.

Delphiniflexine, a new norditerpenoid alkaloid, together with the three known norditerpenoid alkaloids methyllycaconitine, ajadine, and acoseptrigenine were isolated from the aerial parts of Delphinium flexuosum. The structure of delphiniflexine was established on the basis of 1H, 13C, DEPT, homonuclear 1H COSY, NOESY, HSQC, and HMBC NMR studies.

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.

Diterpenoid Alkaloids of Aconitum vulparia Rchb.

From the roots of Aconitum vulparia Rchb., collected in Prüm (Germany), a new norditerpenoid alkaloid, named alexhumboldtine, has been isolated along with the known norditerpenoid alkaloids lappaconitine, anthranoyllycoctonine, lycoctonine, puberaconitine, ajacine, and septentriodine. The structure of alexhumboldtine was established on the basis of 1H, 13C, DEPT, homonuclear 1H COSY, NOESY, HSQC, and HMBC NMR studies. From the aerial parts of the plant another norditerpenoid alkaloid, aconorine, has been isolated.

Observation of intermolecular interactions in large protein complexes by 2D-double difference nuclear Overhauser enhancement spectroscopy: application to the 44 kDa interferon-receptor complex.

NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-α2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.

The effect of ring size variation on the structure and stability of lanthanide(iii) complexes with crown ethers containing picolinate pendants

The coordination properties of the macrocyclic receptor N,N'-bis[(6-carboxy-2-pyridyl)methylene]-1,10-diaza-15-crown-5 (H(2)bp15c5) towards the lanthanide ions are reported. Thermodynamic stability constants were determined by pH-potentiometric titration at 25 °C in 0.1 M KCl. A smooth decrease in complex stability is observed upon decreasing the ionic radius of the Ln(III) ion from La [log K(LaL) = 12.52(2)] to Lu [log K(LuL) = 10.03(6)]. Luminescence lifetime measurements recorded on solutions of the Eu(III) and Tb(III) complexes confirm the absence of inner-sphere water molecules in these complexes. (1)H and (13)C NMR spectra of the complexes formed with the diamagnetic La(III) metal ion were obtained in D(2)O solution and assigned with the aid of HSQC and HMBC 2D heteronuclear experiments, as well as standard 2D homonuclear COSY and NOESY spectra. The (1)H NMR spectra of the paramagnetic Ce(III), Eu(III) and Yb(III) complex suggest nonadentate binding of the ligand to the metal ion. The syn conformation of the ligand in [Ln(bp15c5)](+) complexes implies the occurrence of two helicities, one associated with the layout of the picolinate pendant arms (absolute configuration Δ or Λ), and the other to the five five-membered chelate rings formed by the binding of the crown moiety (absolute configuration δ or λ). A detailed conformational analysis performed with the aid of DFT calculations (B3LYP model) indicates that the complexes adopt a Λ(λδ)(δδλ) [or Δ(δλ)(λλδ)] conformation in aqueous solution. Our calculations show that the interaction between the Ln(III) ion and several donor atoms of the crown moiety is weakened as the ionic radius of the metal ion decreases, in line with the decrease of complex stability observed on proceeding to the right across the lanthanide series.

Intermolecular Interactions in a 44 kDa Interferon−Receptor Complex Detected by Asymmetric Reverse-Protonation and Two-Dimensional NOESY

Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNalpha2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular mass (44 kDa). Sequential and side chain assignment of IFNAR2-EC and IFNalpha2 in their binary complex helped assign the intermolecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC-IFNalpha2 complex was calculated on the basis of the intermolecular interactions found in this study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNalpha2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of intermolecular hydrogen bonds, salt bridges, and van der Waals interactions. Furthermore, our model reveals the participation of several new regions in the binding site such as the N-terminus and A helix of IFNalpha2 and the C domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNalpha2 has changed by 30 degrees in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNalpha2 upon binding of IFNAR1-EC to the binary IFNalpha2-IFNAR2-EC complex.

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

Synthesis, structural and conformational study of some amides derived from 3-methyl-3-azabicyclo[3.2.1]octan-8α(β)-amines

Some amides ( 1α– 7α and 1β– 7β) derived from 3-methyl-3-azabicyclo[3.2.1]octan-8α(β)-amines were synthesized and studied by IR, 1H and 13C NMR spectroscopies. The assignment of all carbon and protons resonances was achieved through the application of one dimensional selective NOE and two dimensional NMR techniques: homonuclear NOESY and heteronuclear 1H– 13C gHSQC correlated spectroscopies. Total correlation spectroscopy (TOCSY) experiments were also carried out. In CDCl 3 solution, at room temperature, all compounds adopt a chair-envelope conformation with the N–CH 3 group in equatorial disposition. In the α-epimers the piperidine ring is puckered at C8 to relieve the interactions between the amido group and the H6(7)x protons. α- and β-Epimers show a preferred trans disposition for the NH–CO group and free rotation of the NH–CO–R group around the C8–NH bond. Finally, NMR and IR data reveal that compounds 7α and 7β adopt in CDCl 3 solution a preferred s-cis conformation for the O C–C C system, the proportion of this conformation increasing when the polarity of the solvent decreases.

Structural Analysis of Conserved Oligomeric Golgi Complex Subunit 2

The conserved oligomeric Golgi (COG) complex is strongly implicated in retrograde vesicular trafficking within the Golgi apparatus. Although its mechanism of action is poorly understood, it has been proposed to function by mediating the initial physical contact between transport vesicles and their membrane targets. An analogous role in tethering vesicles has been suggested for at least six additional large multisubunit complexes, including the exocyst, a complex essential for trafficking to the plasma membrane. Here we report the solution structure of a large portion of yeast Cog2p, one of eight subunits composing the COG complex. The structure reveals a six-helix bundle with few conserved surface features but a general resemblance to recently determined crystal structures of four different exocyst subunits. This finding provides the first structural evidence that COG, like the exocyst and potentially other tethering complexes, is constructed from helical bundles. These structures may represent platforms for interaction with other trafficking proteins including SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) and Rabs.

Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

Structure of FBP11 WW1-PL Ligand Complex Reveals the Mechanism of Proline-rich Ligand Recognition by Group II/III WW Domains

FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.

AUREMOL-RFAC-3D, combination of R-factors and their use for automated quality assessment of protein solution structures

We present here the computer program AUREMOL-RFAC-3D that is a generalization of the previously published program RFAC for the fully automated estimation of residual indices (R-factors) from 2D NOESY spectra. It is part of the larger AUREMOL software package (www.auremol.de). RFAC-3D calculates R-factors directly from two-dimensional homonuclear NOESY spectra as well as from three-dimensional (15)N or (13)C edited NOESY-HSQC spectra and thus extends the application range to larger proteins. The fully automated method includes automated peak picking and integration, a Bayesian noise and artifact recognition and the use of the complete relaxation matrix formalism. To enhance the reliability of the calculated R-factors the method is also generalized to calculate combined R-factors from a set of 2D and 3D-spectra. For an optimal combination of the information derived from different sources a plausible formalism had to be derived. In addition, we present a novel direct R-factors based measure that correlates an R-factors as defined in this paper to the root mean square deviation of the actual structure from the optimal structure. The new program has been successfully tested on the histidine containing phosphocarrier protein (HPr) from Staphylococcus carnosus and on the Ras-binding domain (RBD) of the Ral guanine-nucleotide dissociation stimulation factor (RalGDS).

Comparison of Lipoteichoic Acid from Different Serotypes of Streptococcus pneumoniae

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.