Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli.

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope ß is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a ß epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Molecular organization of various collagen fragments as revealed by atomic force microscopy and diffusion-ordered NMR spectroscopy.

Heterogeneous mixtures of collagen fragments can be used as nutrition supplement or as key ingredients for ointments with therapeutic relevance in wound healing. Some mixtures of collagen fragments are referred to as collagen hydrolysates owing to the production process with hydrolytic enzymes. Since the precise composition of collagen hydrolysates is generally unknown, it is of interest to analyze samples containing various collagen fragments with appropriate biophysical methods. Any product optimization without a profound knowledge concerning the size and the molecular weight distribution of its components is nearly impossible. It turned out that a combination of AFM methods with NMR techniques is exceptionally suited to examine the size range and the aggregation behavior of the collagen fragments in the hydrolysates of fish, jellyfish, chicken, porcine and bovine collagen. Supported by molecular modeling calculations, the AFM and NMR experiments provide a detailed knowledge about the composition of collagen hydrolysates and collagen ointments. Furthermore, the data allow a correlation between the size of the fragments and their potential bioactivity.

R Shiny App for the Automated Deconvolution of NMR Spectra to Quantify the Solid-State Forms of Pharmaceutical Mixtures.

Bioavailability and chemical stability are important characteristics of drug products that are strongly affected by the solid-state structure of the active pharmaceutical ingredient (API). In pharmaceutical development and quality control activities, solid-state NMR (ssNMR) has proved to be an excellent tool for the detection and accurate quantification of undesired solid-state forms. To obtain correct quantitative outcomes, the resulting spectrum of an analytical sample should be deconvoluted into the individual spectra of the pure components. However, the ssNMR deconvolution is particularly challenging due to the following: the relatively large line widths that may lead to severe peak overlap, multiple spinning sidebands as a result of applying Magic Angle Spinning (MAS), and highly irregular peak shapes commonly observed in mixture spectra. To address these challenges, we created a tailored and automated deconvolution approach of ssNMR mixture spectra that involves a linear combination modelling (LCM) of previously acquired reference spectra of pure solid-state components. For optimal model performance, the template and mixture spectra should be acquired under the same conditions and experimental settings. In addition to the parameters controlling the contributions of the components in the mixture, the proposed model includes terms for spectral processing such as phase correction and horizontal shifting that are all jointly estimated via a non-linear, constrained optimisation algorithm. Finally, our novel procedure has been implemented in a fully functional and user-friendly R Shiny webtool (hence no local R installation required) that offers interactive data visualisations, manual adjustments to the automated deconvolution results, and the traceability and reproducibility of analyses.

Elucidation of lignin structure by quantitative 2D NMR.

Quick quantitative HSQC (QQ-HSQC) was applied to quantitative evaluation of different inter-unit linkages in an array of milled softwood and hardwood and technical lignins by using the guaiacyl C2 and syringyl C2-C6 signals as internal standards. The results were found to be highly reproducible and comparable with earlier literature reports. The advantage of QQ-HSQC NMR analysis of lignin is contemporary detection and quantification of lignin inter-unit linkages with a direct, non-destructive method requiring short acquisition times.

Practical synthesis of (2'R)-2'-deoxy-2'-C-methyluridine by highly diastereoselective homogeneous hydrogenation.

Diastereoselective hydrogenation of 2'-deoxy-2'-exo-methyleneuridine was carried out under homogeneous conditions using a low loading of a chiral Rh catalyst. This, coupled with improvements in the synthesis of the substrate, allowed the smooth pilot plant preparation of the title compound on >10 kg scale.

Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor.

Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.

Why structurally different cyclic peptides can be glycomimetics of the HNK-1 carbohydrate antigen.

The cyclic peptides c-(LSETTl) and c-(RTLPFS) are of potential clinical interest--they stimulate neurite outgrowth in a way that is similar to the effects of the HNK-1 (human natural killer cell-1) antigenic carbohydrate chains, which are terminated by 3'-sulfated glucuronic acid attached to an N-acetyllactosamine unit. To investigate the structure-activity relationships of the ability of the cyclic peptides to mimic HNK-1 carbohydrates, conformational analysis and examination of hydrophobic and hydrophilic patterns were performed and compared with the characteristics of a synthetic HNK-1 trisaccharide derivative. Data obtained demonstrate that both the trisaccharide and the glycomimetic peptide c-(LSETTl) exhibit a similar relationship between their hydrophobic moieties and their negatively charged sites. However, the second cyclic glycomimetic peptide investigated here, c-(RTLPFS), has a positively charged group as a potential contact point due to its Arg residue. Therefore, we studied the amino acid composition of all known receptor structures in the Protein Data Bank that are in contact with uronic acid and/or sulfated glycans. Interactions of the HNK-1 trisaccharide, c-(LSETTl), and c-(RTLPFS) with a laminin fragment involved in HNK-1 carbohydrate binding (i.e., the 21mer peptide: KGVSSRSYVGCIKNLEISRST) were also analyzed. Because the structure of the HNK-1-binding laminin domain is not available in the Protein Data Bank, we used the HNK-1-binding 21mer peptide fragment of laminin for the construction of a model receptor that enabled us to compare the molecular interplay of the HNK-1 trisaccharide and the two cyclopeptides c-(LSETTl) and c-(RTLPFS) with a reliable receptor structure in considerable detail.

Strategies for Identity Testing of Therapeutic Oligonucleotide Drug Substances and Drug Products.

A risk-based approach for routine identity testing of therapeutic oligonucleotide drug substances and drug products is described. Risk analysis of solid-phase oligonucleotide synthesis indicates that intact mass measurement is a powerful technique for confirming synthesis of the intended oligonucleotide. Further risk assessment suggests that the addition of a second, sequence-sensitive identity test, which relies on a comparison of some property of the sample to a reference standard of proven identity, results in a sufficient test of identity for most oligonucleotide drug substances and products. Alternative strategies for drug product identity testing are presented. The analysis creates a common way to communicate risk and should result in a harmonized approach to identity testing that avoids the unnecessary analytical burden associated with routine de novo sequencing, without compromising quality or patient safety.

The tandem zinc-finger region of human ZHX adopts a novel C2H2 zinc finger structure with a C-terminal extension.

Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation.

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis.

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni alpha-(2-->3)-sialyltransferase (CST-06) and beta-(1-->4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C-1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented.

Gradual phosphorylation regulates PC4 coactivator function.

The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces.

An alternate conformation of the hyperthermostable HU protein from Thermotoga maritima has unexpectedly high flexibility.

The homodimeric HU protein from the hyperthermophile Thermotoga maritima (HUTmar) is a model system which can yield insights into the molecular determinants of thermostability in proteins. Unusually for a thermostable protein, HUTmar exists in a structurally heterogeneous state as evidenced by the assignment of two distinct and approximately equally populated forms in solution. Relaxation measurements combined with chemical shift, hydrogen exchange, and nuclear Overhauser enhancement data confirm the main structural features of both forms. In addition, these data support a two-state model for HUTmar in which the major form closely resembles the X-ray structure while the very flexible minor form is less structured. HUTmar may therefore be a new example of the small class of hyperthermostable proteins with unexpected flexibility.

Mechanism of 3D domain swapping in bovine seminal ribonuclease.

3D domain swapping (3D-DS) is a complex protein aggregation process for which no unique mechanism exists. We report an analysis of 3D-DS in bovine seminal ribonuclease, a homodimeric protein whose subunits are linked by two disulfide bridges, based on NMR and biochemical studies. The presence of the covalent bonds between the subunits stabilizes the unswapped dimer, and allows distinct evaluation of the structural and dynamic effects of the swapping with respect to the dimerization process. In comparison with the monomeric subunit, which, in solution has a compact structure without any propensity for local unfolding, both swapped and unswapped dimers show increased flexibility. NMR analysis, together with urea denaturation and hydrogen­deuterium exchange data, indicates that the two dimers have increased conformational fluctuations. Furthermore, we found that the rate-limiting step of both the swapping and unswapping pathways is the detachment of the N-terminal helices from the monomers. These results suggest a new general mechanism in which a dimeric intermediate could facilitate 3D-DS in globular proteins.

NMR studies on structure and dynamics of the monomeric derivative of BS-RNase: new insights for 3D domain swapping.

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

Interaction of the α2A domain of integrin with small collagen fragments.

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.

WITHDRAWN: Corrigendum to "A lectin from the Chinese bird-hunting spider binds sialic acids"

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi: 10.1016/j.carres.2010.01.003. The duplicate article has therefore been withdrawn.

A lectin from the Chinese bird-hunting spider binds sialic acids.

The affinity to sialic acid-containing oligosaccharides of the small-animal lectin SHL-I isolated from the venom of the Chinese bird-hunting spider Selenocosmia huwena is here described for the first time. By a strategic combination of NMR techniques, molecular modeling, and data mining tools it was possible to identify the crucial amino acid residues that are responsible for SHL-I's ability to bind sialic acid residues in a specific way. Furthermore, we are able to discuss the role of the functional groups of sialic acid when bound to SHL-I. Also the impact of Pro31 in its cis- or trans-form on SHL-I's ligand affinity is of special interest, since it answers the question if Trp32 is a crucial amino acid for stabilizing complexes between SHL-I and sialic acid. SHL-I can be considered as a proper model system that provides further insights into the binding mechanisms of small-animal lectins to sialic acid on a sub-molecular level.

Structural dynamics in the activation of Epac.

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.