Structural Insight into the Binding of TGIF1 to SIN3A PAH2 Domain through a C-Terminal Amphipathic Helix

TGIF1 is a transcriptional repressor playing crucial roles in human development and function and is associated with holoprosencephaly and various cancers. TGIF1-directed transcriptional repression of specific genes depends on the recruitment of corepressor SIN3A. However, to date, the exact region of TGIF1 binding to SIN3A was not clear, and the structural basis for the binding was unknown. Here, we demonstrate that TGIF1 utilizes a C-terminal domain (termed as SIN3A-interacting domain, SID) to bind with SIN3A PAH2. The TGIF1 SID adopts a disordered structure at the apo state but forms an amphipathic helix binding into the hydrophobic cleft of SIN3A PAH2 through the nonpolar side at the holo state. Residues F379, L382 and V383 of TGIF1 buried in the hydrophobic core of the complex are critical for the binding. Moreover, homodimerization of TGIF1 through the SID and key residues of F379, L382 and V383 was evidenced, which suggests a dual role of TGIF1 SID and a correlation between dimerization and SIN3A-PAH2 binding. This study provides a structural insight into the binding of TGIF1 with SIN3A, improves the knowledge of the structure–function relationship of TGIF1 and its homologs and will help in recognizing an undiscovered SIN3A-PAH2 binder and developing a peptide inhibitor for cancer treatment.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Structure of the human ATM kinase and mechanism of Nbs1 binding

DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and ultimately cancer. Central to the sensing of DSBs are ATM (Ataxia telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, and the MRN (Mre11-Rad50-Nbs1) protein complex that activates ATM. How the MRN complex recruits and activates ATM kinase is poorly understood. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM kinase, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped autoinhibited structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these interactions with mutagenesis and biochemical assays.

Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k

Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

Identification of novel thiadiazin derivatives as potentially selective inhibitors towards trypanothione reductase from Trypanosoma cruzi by molecular docking using the numerical index poses ratio Pr and the binding mode analysis

Chagas disease is a serious health problem in Central and South America for which effective treatment is not currently available. This illness is caused by the protozoa Trypanosoma cruzi, a species that relies on a thiol-based metabolism to regulate oxidative stress. Trypanothione reductase enzyme plays a central role in the metabolic pathway of the parasite. In this work, a virtual screening of a library of novel thiadiazine derivatives against trypanothione reductase using molecular docking was performed. Four different series of hybrid ligands having in the structure one or two peptoid moieties (series I and II) or the tetrazole ring (series III and IV) were considered. An ad hoc numerical index called poses ratio was introduced to interpret the results of the docking analysis and to establish relevant structure-interaction relationships. In addition, six binding modes were found for the ligands with the highest populated conformational clusters after applying contact-based analysis. The most regular and relevant were binding modes I and II, found mainly for ligands from series I. A subsequent molecular docking on human glutathione reductase enzyme allowed to assess the possible cytotoxicity of the ligands towards human cells. A selective binding profile was found for ligands with interactions in the Hydrophobic cleft, the spermidine and the Z subsites inside the active site of trypanothione reductase. At the end of the study, new thiadiazine-based compounds were identified as plausible candidates to selectively inhibit the parasitic enzyme. Graphic abstract

2-(2-Methyl-2-nitrovinyl)furan but Not Furvina Interfere with Staphylococcus aureus Agr Quorum-Sensing System and Potentiate the Action of Fusidic Acid against Biofilms

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1–4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant

Background. SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA Polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. Methods. We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance.Results. We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed that the 14408 mutation, emerged for the first time in Europe in mid-February 2020, is present in the SARS-CoV-2 RdRp gene sequence. Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). Conclusions. These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.

Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1–121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50–57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.