Master corepressor inactivation through multivalent SLiM-induced polymerization mediated by the oncogene suppressor RAI2.

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.

Backbone and side chain resonance assignment of the intrinsically disordered human DBNDD1 protein.

The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein.

NMR of intrinsically disordered proteins: A note on the application of 15N-13Cα het-TOCSY mixing for 13Cα magnetisation transfers.

Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue 'i' to 'i + 1' and 'i - 1' residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.

Backbone and nearly complete side-chain chemical shift assignments reveal the human uncharacterized protein CXorf51A as intrinsically disordered.

Even though the human genome project showed that our DNA contains a mere 20,000 to 25,000 protein coding genes, an unexpectedly large number of these proteins remain functionally uncharacterized. A structural characterization of these "unknown" proteins may help to identify possible cellular tasks. We therefore used a combination of bioinformatics and nuclear magnetic resonance spectroscopy to structurally de-orphanize one of these gene products, the 108 amino acid human uncharacterized protein CXorf51A. Both our bioinformatics analysis as well as the [Formula: see text]H, [Formula: see text]C, [Formula: see text]N backbone and near-complete side-chain chemical shift assignments indicate that it is an intrinsically disordered protein.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Backbone and nearly complete side-chain chemical shift assignments of the human death-associated protein 1 (DAP1).

Death-associated protein 1 (DAP1) is a proline-rich cytoplasmatic protein highly conserved in most eukaryotes. It has been reported to be involved in controlling cell growth and migration, autophagy and apoptosis. The presence of human DAP1 is associated to a favourable prognosis in different types of cancer. Here we describe the almost complete [Formula: see text], [Formula: see text], and [Formula: see text] chemical shift assignments of the human DAP1. The limited spectral dispersion, mainly in the [Formula: see text] region, and the lack of defined secondary structure elements, predicted based on chemical shifts, identifies human DAP1 as an intrinsically disordered protein (IDP). This work lays the foundation for further structural investigations, dynamic studies, mapping of potential interaction partners or drug screening and development.

1H, 13C, and 15N backbone assignments of the C-terminal region of the human retinoic acid-induced protein 2.

Retinoic acid-induced protein 2 is a human protein of 530 residues encoded by the RAI2 gene (Q9Y5P3; RAI2_HUMAN). RAI2 is a novel tumor suppressor protein whose depletion in breast cancer cell lines results in the downregulation of several genes associated with differentiation along with increased invasiveness and aggressive tumor phenotype of the cells. The role of the protein is specified to be a transcriptional regulator that promotes chromosomal stability and hence controls the expression of several regulators of cancer and metastasis. Structurally, RAI2 remains an unknown entity and, hence, to obtain a detailed view on the structure function relationship we report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of the C-terminal region (a.a. 303-451 of UniProt Q9Y5P3) of RAI2.

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR.

Disulfide bridges establish a fundamental element in the molecular architecture of proteins and peptides which are involved e.g., in basic biological processes or acting as toxins. NMR spectroscopy is one method to characterize the structure of bioactive compounds including cystine-containing molecules. Although the disulfide bridge itself is invisible in NMR, constraints obtained via the neighboring NMR-active nuclei allow to define the underlying conformation and thereby to resolve their functional background. In this mini-review we present shortly the impact of cysteine and disulfide bonds in the proteasome from different domains of life and give a condensed overview of recent NMR applications for the characterization of disulfide-bond containing biomolecules including advantages and limitations of the different approaches.

1H, 13C, and 15N Backbone assignments of the human brain and acute leukemia cytoplasmic (BAALC) protein.

The brain and acute leukemia cytoplasmic (BAALC; UniProt entry Q8WXS3) is a 180-residue-long human protein having six known isoforms. BAALC is expressed in either hematopoietic or neuroectodermal cells and its specific function is still to be revealed. However, as a presumably membrane-anchored protein at the cytoplasmic side it is speculated that BAALC exerts its function at the postsynaptic densities of certain neurons and might play a role in developing cytogenetically normal acute myeloid leukemia (CN-AML) when it is highly overexpressed by myeloid or lymphoid progenitor cells. In order to better understand the physiological role of BAALC and to provide the basis for a further molecular characterization of BAALC, we report here the 1H, 13C, and 15N resonance assignments for the backbone nuclei of its longest hematopoietic isoform (isoform 1). In addition, we present a 1HN and 15NH chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the 1H and 15N chemical shifts.

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP.

BACKGROUND & MOTIVATION: Peptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid ß, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme. METHODS: We employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes. RESULTS: Heme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly. CONCLUSIONS: We identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity. GENERAL SIGNIFICANCE: The identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.

Structure of an RNA aptamer in complex with the fluorophore tetramethylrhodamine.

RNA aptamers-artificially created RNAs with high affinity and selectivity for their target ligand generated from random sequence pools-are versatile tools in the fields of biotechnology and medicine. On a more fundamental level, they also further our general understanding of RNA-ligand interactions e. g. in regard to the relationship between structural complexity and ligand affinity and specificity, RNA structure and RNA folding. Detailed structural knowledge on a wide range of aptamer-ligand complexes is required to further our understanding of RNA-ligand interactions. Here, we present the atomic resolution structure of an RNA-aptamer binding to the fluorescent xanthene dye tetramethylrhodamine. The high resolution structure, solved by NMR-spectroscopy in solution, reveals binding features both common and different from the binding mode of other aptamers with affinity for ligands carrying planar aromatic ring systems such as the malachite green aptamer which binds to the tetramethylrhodamine related dye malachite green or the flavin mononucleotide aptamer.

Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation.

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.

Structural insights into heme binding to IL-36α proinflammatory cytokine.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-ß-synthase with heme.

The N-terminal segment of human cystathionine-ß-synthase (CBS(1-40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [1H, 15N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys15-Pro16 motif of CBS(1-40).

1H, 13C, and 15N resonance assignments of the cytokine interleukin-36ß isoform-2.

Interleukins are cytokines performing central tasks in the human immune system. Interleukin-36ß (IL-36ß) is a member of the interleukin-1 superfamily as are its homologues IL-36α and IL-36γ. All of them interact with a common receptor composed of IL-36R and IL-1R/acP. IL-36 cytokines can activate IL-36R to proliferation of CD4 + lymphocytes or stimulate M2 macrophages as potently as IL-1ß. Within our efforts to study the structure-function relationship of the three interleukins IL-36α, IL-36ß and IL-36γ by heteronuclear multidimensional NMR, we here report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of cytokine interleukin-36ß isoform-2.

Conformational Plasticity in Broadly Neutralizing HIV-1 Antibodies Triggers Polyreactivity.

Human high-affinity antibodies to pathogens often recognize unrelated ligands. The molecular origin and the role of this polyreactivity are largely unknown. Here, we report that HIV-1 broadly neutralizing antibodies (bNAbs) are frequently polyreactive, cross-reacting with non-HIV-1 molecules, including self-antigens. Mutating bNAb genes to increase HIV-1 binding and neutralization also results in de novo polyreactivity. Unliganded paratopes of polyreactive bNAbs with improved HIV-1 neutralization exhibit a conformational flexibility, which contributes to enhanced affinity of bNAbs to various HIV-1 envelope glycoproteins and non-HIV antigens. Binding adaptation of polyreactive bNAbs to the divergent ligands mainly involves hydrophophic interactions. Plasticity of bNAbs' paratopes may, therefore, facilitate accommodating divergent viral variants, but it simultaneously triggers promiscuous binding to non-HIV-1 antigens. Thus, a certain level of polyreactivity can be a mark of adaptable antibodies displaying optimal pathogens' recognition.

Synthesis and biochemical evaluation of two novel N-hydroxyalkylated cyclosporin A analogs.

The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.

Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-ß-synthase.

Cystathionine-ß-synthase (CBS) belongs to a large family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1-40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Here we report the NMR resonance assignment and heme interaction studies for the N-terminal peptide stretch of CBS. We present NMR-spectral evidence that residues 1-40 constitute an intrinsically disordered region in CBS and interact with heme via a cysteine-proline based motif.

Conformational µ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide µ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual 1H and 13C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.