Oliver Ohlenschläger

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.

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. The specific properties of these repeated interactions open up new therapeutic opportunities.

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.

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.

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.

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.

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 ¹H^N and ¹⁵N^H chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the ¹H and ¹⁵N chemical shifts.