1H, 13C and 15N backbone and side-chain resonance assignments of the human oncogenic protein NCYM.

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1H, 15N and 13C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners.

Combining High-Pressure NMR and Geometrical Sampling to Obtain a Full Topological Description of Protein Folding Landscapes: Application to the Folding of Two MAX Effectors from Magnaporthe oryzae.

Despite advances in experimental and computational methods, the mechanisms by which an unstructured polypeptide chain regains its unique three-dimensional structure remains one of the main puzzling questions in biology. Single-molecule techniques, ultra-fast perturbation and detection approaches and improvement in all-atom and coarse-grained simulation methods have greatly deepened our understanding of protein folding and the effects of environmental factors on folding landscape. However, a major challenge remains the detailed characterization of the protein folding landscape. Here, we used high hydrostatic pressure 2D NMR spectroscopy to obtain high-resolution experimental structural information in a site-specific manner across the polypeptide sequence and along the folding reaction coordinate. We used this residue-specific information to constrain Cyana3 calculations, in order to obtain a topological description of the entire folding landscape. This approach was used to describe the conformers populating the folding landscape of two small globular proteins, AVR-Pia and AVR-Pib, that belong to the structurally conserved but sequence-unrelated MAX effectors superfamily. Comparing the two folding landscapes, we found that, in spite of their divergent sequences, the folding pathway of these two proteins involves a similar, inescapable, folding intermediate, even if, statistically, the routes used are different.

Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds.

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution.

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)'s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis.

Tuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.

A Tumor-Imaging Method Targeting Cancer-Associated Fibroblasts.

The tumor stroma, which accounts for a large part of the tumor mass, represents an attractive target for the delivery of diagnostic and therapeutic compounds. Here, the focus is notably on a subpopulation of stromal cells, known as cancer-associated fibroblasts, which are present in more than 90% of epithelial carcinomas, including pancreatic, colon, and breast cancer. Cancer-associated fibroblasts feature high expression of fibroblast activation protein (FAP), which is not detectable in adult normal tissue but is associated with a poor prognosis in cancer patients. Methods: We developed an iodinated and a DOTA-coupled radiotracer based on a FAP-specific enzyme inhibitor (FAPI) and evaluated them in vitro using uptake, competition, and efflux studies as well as confocal microscopy of a fluorescence-labeled variant. Furthermore, we performed imaging and biodistribution studies on tumor-bearing animals. Finally, proof of concept was realized by imaging patients with 68Ga-labeled FAPI. Results: Both FAPIs showed high specificity, affinity, and rapid internalization into FAP-expressing cells in vitro and in vivo. Biodistribution studies on tumor-bearing mice and on the first cancer patients demonstrated high intratumoral uptake of the tracer and fast body clearance, resulting in high-contrast images and negligible exposure of healthy tissue to radiation. A comparison with the commonly used radiotracer 18F-FDG in a patient with locally advanced lung adenocarcinoma revealed that the new FAP ligand was clearly superior. Conclusion: Radiolabeled FAPIs allow fast imaging with very high contrast in tumors having a high stromal content and may therefore serve as pantumor agents. Coupling of these molecules to DOTA or other chelators allows labeling not only with 68Ga but also with therapeutic isotopes such as 177Lu or 90Y.

Combination of phage display and molecular grafting generates highly specific tumor-targeting miniproteins.

Frankenstein's peptide: the grafting of the binding domain from miniprotein Min-23 into the sunflower trypsin inhibitor (SFTI-I) peptide scaffold preserved its in vitro and in vivo binding specificity and proteolytic stability. The combination of these peptides was shown to be tumor-specific with a good binding affinity for delta-like ligand 4 (Dll4) protein. The use of SFTI-I as a peptide scaffold is ideal for hit-to-lead development.

Dynamic and structural characterization of a bacterial FHA protein reveals a new autoinhibition mechanism.

The OdhI protein is key regulator of the TCA cycle in Corynebacterium glutamicum. This highly conserved protein is found in GC rich Gram-positive bacteria (e.g., the pathogenic Mycobacterium tuberculosis). The unphosphorylated form of OdhI inhibits the OdhA protein, a key enzyme of the TCA cycle, whereas the phosphorylated form is inactive. OdhI is predicted to be mainly a single FHA domain, a module that mediates protein-protein interaction through binding of phosphothreonine peptides, with a disordered N-terminal extension substrate of the serine/threonine protein kinases. In this study, we solved the solution structure of the unphosphorylated and phosphorylated isoforms of the protein. We observed a major conformational change between the two forms characterized by the binding of the phosphorylated N-terminal part of the protein to its own FHA domain, consequently inhibiting it. This structural observation corresponds to a new autoinhibition mechanism described for a FHA domain protein.