The ALS drug riluzole binds to the C-terminal domain of SARS-CoV-2 nucleocapsid protein and has antiviral activity.

Nucleoproteins (N) play an essential role in virus assembly and are less prone to mutation than other viral structural proteins, making them attractive targets for drug discovery. Using an NMR fragment-based drug discovery approach, we identified the 1,3-benzothiazol-2-amine (BZT) group as a scaffold to develop potential antivirals for SARS-CoV-2 nucleocapsid (N) protein. A thorough characterization of BZT derivatives using NMR, X-ray crystallography, antiviral activity assays, and intrinsic fluorescence measurements revealed their binding in the C-terminal domain (CTD) domain of the N protein, to residues Arg 259, Trp 330, and Lys 338, coinciding with the nucleotide binding site.

Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging.

Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis.

The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner.

The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe.

Structural basis of Nrd1-Nab3 heterodimerization.

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID).

Urine NMR-based TB metabolic fingerprinting for the diagnosis of TB in children.

Tuberculosis (TB) is a major cause of morbidity and mortality in children, and early diagnosis and treatment are crucial to reduce long-term morbidity and mortality. In this study, we explore whether urine nuclear magnetic resonance (NMR)-based metabolomics could be used to identify differences in the metabolic response of children with different diagnostic certainty of TB. We included 62 children with signs and symptoms of TB and 55 apparently healthy children.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition.

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain.

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death.

Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive.

Interaction Networks in Protein Folding via Atomic-Resolution Experiments and Long-Time-Scale Molecular Dynamics Simulations.

The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue-residue couplings associated with cooperative folding emerges.

Two singular types of CCCH tandem zinc finger in Nab2p contribute to polyadenosine RNA recognition.

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 μM).

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF-VEGFR interaction.

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages.

Nuclear magnetic resonance study of protein-protein interactions involving apoptosis regulator Diva (Boo) and the BH3 domain of proapoptotic Bcl-2 members.

According to biochemical assays, the Bcl-2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease-activating factor Apaf-1. In typical Bcl-2 heterodimers, peptide fragments comprising the Bcl-2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High-resolution structural studies have revealed that the BH3 peptide forms an α-helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein.

Cyclic amino acid linkers stabilizing key loops of brain derived neurotrophic factor.

Based on β-turn-like BDNF loops 2 and 4, involved in receptor interaction, cyclic peptide replicas were designed, synthesized and tested. In addition to the native turn residues, the cyclic peptides include a linker unit between the N- and C-termini, selected by molecular modeling among various non-proteinogenic cyclic amino acids. NMR conformational studies showed that most of the cyclic peptides were able to adopt turn-like structures.

Pub1p C-terminal RRM domain interacts with Tif4631p through a conserved region neighbouring the Pab1p binding site.

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion.

Trp-Trp pairs as β-hairpin stabilisers: hydrogen-bonded versus non-hydrogen-bonded sites.

Trp-Trp pairs have emerged as a successful strategy for β-hairpin stabilization. Using loop 3 of Vammin as a template, we experimentally demonstrate that the contribution of Trp-Trp pairs to β-hairpin stability depends on β-sheet periodicity, that is, they are stabilising at non-hydrogen-bonded sites, but not at hydrogen-bonded positions.

Structural autonomy of a β-hairpin peptide derived from the pneumococcal choline-binding protein LytA.

The cell wall of Streptococcus pneumoniae and several other micro-organisms is decorated with a number of the so-called choline-binding proteins (CBPs) that recognise the choline residues in the bacterial surface by means of highly conserved, concatenated 20-aa sequences termed choline-binding repeats (CBRs), that are composed of a loop and a β-hairpin structure. In this work, we have investigated the ability to fold in aqueous solution of a 14-aa peptide (LytA₁₉₇₋₂₁₀[wt]) and a single derivative of it, LytA₁₉₇₋₂₁₀[ND], corresponding to one of the six β-hairpins of the LytA pneumococcal amidase. Intrinsic fluorescence and circular dichroism spectroscopical measurements showed that both peptides spontaneously acquire a non-random conformation which is also able to bind the natural ligand choline.

Tryptophan residues: scarce in proteins but strong stabilizers of β-hairpin peptides.

Tryptophan plays important roles in protein stability and recognition despite its scarcity in proteins. Except as fluorescent groups, they have been used rarely in peptide design. Nevertheless, Trp residues were crucial for the stability of some designed minimal proteins.

Sequence inversion and phenylalanine surrogates at the beta-turn enhance the antibiotic activity of gramicidin S.

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles.

A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix.

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor-antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter.

Molecular basis of histone H3K36me3 recognition by the PWWP domain of Brpf1.

Trimethylation of Lys36 in histone H3 (H3K36me3) coordinates events associated with the elongation phase of transcription and is also emerging as an important epigenetic regulator of cell growth and differentiation. We have identified the PWWP domain of bromo and plant homeodomain (PHD) finger-containing protein 1 (BRPF1) as a H3K36me3 binding module and have determined the structure of this domain in complex with an H3K36me3-derived peptide.

1H, 13C and 15N backbone and side chain resonance assignments of the C-terminal domain of CdnL from Myxococcus xanthus.

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the (1)H, (13)C and (15)N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.