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Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of anHDAC2knockoutin vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators.GRAPHICAL ABSTRACT

The fluorine-19 nucleus was recognized early to harbor exceptional properties for NMR spectroscopy. With 100% natural abundance, a high gyromagnetic ratio (83% sensitivity compared to (1)H), a chemical shift that is extremely sensitive to its surroundings and near total absence in biological systems, it was destined to become a favored NMR probe, decorating small and large molecules. However, after early excitement, where uptake of fluorinated aromatic amino acids was explored in a series of animal studies, (19)F-NMR lost popularity, especially in large molecular weight systems, due to chemical shift anisotropy (CSA) induced line broadening at high magnetic fields. Recently, two orthogonal approaches, i) CF(3) labeling and ii) aromatic (19)F-(13)C labeling leveraging the TROSY (Transverse Relaxation Optimized Spectroscopy) effect have been successfully applied to study large biomolecular systems. In this perspective, we will discuss the fascinating early work with fluorinated aromatic amino acids, which reveals the enormous potential of these non-natural amino acids in biological NMR and the potential of (19)F-NMR to characterize protein and nucleic acid structure, function and dynamics in the light of recent developments. Finally, we explore how fluorine NMR might be exploited to implement small molecule or fragment screens that resemble physiological conditions and discuss the opportunity to follow the fate of small molecules in living cells.

Summary The target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes., Graphical abstract, Highlights • The S.pombe Folliculin complex, Bhd1-Fnp1 Complex (BFC) augments repression or activation of TORC1 in response to amino acid levels, similar to a rheostat • BFC interacts and regulates V-ATPase activity in response to amino acid levels • Proteomic data reveal Ptr2 and Ade3 as novel amino acid-dependent regulators of TORC1 • S.pombe is an excellent model for studying the tumor suppressor function of BFC and related proteins, Cellular physiology; Cell biology; Functional aspects of cell biology

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here, we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites’ life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5′ mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

On average, an approved drug currently costs US$2–3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins. VirtualFlow, an open-source drug discovery platform, enables the efficient preparation and virtual screening of ultra-large ligand libraries to identify molecules that bind with high affinity to target proteins.

The Bloch–Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch–Siegert shifts. This approach enables accurate observation of the resonance frequencies of decoupled nuclear spins. We apply this method to increase the resolution of the HNCA experiment. We also observe a doubling in sensitivity for a 30 kDa protein. We demonstrate the use of band-selective Cβ decoupling to produce amino acid-specific line shapes, which are valuable for assigning resonances to the protein sequence. Finally, we assign the backbone of a 30 kDa protein, Human Carbonic Anhydrase II, using only HNCA experiments acquired with band-selective decoupling schemes, and instrument time of one week., Bloch–Siegert shifts prevent the accurate observation of resonance frequencies in NMR experiments. Here the authors present a method for homonuclear decoupling that avoids inducing Bloch–Siegert shifts and improves the sensitivity and resolution of HNCA experiments.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1 H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15 N-detected experiments that take advantage of the slower transverse relaxation property of 15 N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1 H and 13 C resonances. The six 3D experiments described here start with aliphatic 1 H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131–294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine–proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment., Structure determination of large proteins by solution state NMR is challenging due to spectral overlap. Here the authors present a labeling strategy using 2-13C and 3-13C pyruvate as carbon source for E. coli, which increases the effective resolution of triple-resonance HNCA experiments and helps to overcome this problem.

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)4LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)4LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP144-87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m7GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

Purpose of reviewComparative gene identification-58 (CGI-58) is an important player in lipid metabolism. It acts as activator of triglyceride hydrolases and as acyl-CoA-dependent lysophosphatidic acid acyltransferase. This review aims at establishing a structure–function relationship of this still r

Summary Initial molecular details of cellular activation following αβT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3eγ plus CD3eδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αβTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαβ and CD3 associations.

For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the (1)H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording (13)C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon-carbon (C-C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C-C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate (13)C labeling using 2-(13)C glycerol, 2-(13)C pyruvate, or 3-(13)C pyruvate as the carbon source. With the elimination of the strong one-bond C-C coupling, the TROSY effect can easily be exploited. We show that (1)H-(13)C TROSY spectra of alternately (13)C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex.

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS(239)S(240), we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno-blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.

During mitosis, kinetochores coordinate the attachment of centromeric DNA to the dynamic plus ends of microtubules, which is hypothesized to pull sister chromatids toward opposing poles of the mitotic spindle. The outer kinetochore Ndc80 complex acts synergistically with the Ska (spindle and kinetochore-associated) complex to harness the energy of depolymerizing microtubules and power chromosome movement. The Ska complex is a hexamer consisting of two copies of the proteins Ska1, Ska2 and Ska3, respectively. The C-terminal domain of the spindle and kinetochore-associated protein 1 (Ska1) is the microtubule-binding domain of the Ska complex. We solved the solution structure of the C. elegans microtubule-binding domain (MTBD) of the protein Ska1 using NMR spectroscopy. Here, we report the resonance assignments of the MTBD of C. elegans Ska1., Austrian Science Fund (project P22170, and the doctoral school ‘‘DK Molecular Enzymology’’ (W901-B05))

SummaryTo ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule-binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated.

The standard codon table is a primary tool for basic understanding of molecular biology. In the minds of many, the table’s orderly arrangement of bases and amino acids is synonymous with the true genetic code, i.e., the biological coding principle itself. However, developments in the field reveal a much more complex and interesting picture. In this article, we review the traditional codon table and its limitations in light of the true complexity of the genetic code. We suggest the codon table be brought up to date and, as a step, we present a novel superposition of the BLOSUM62 matrix and an allowed point mutation matrix. This superposition depicts an important aspect of the true genetic code—its ability to tolerate mutations and mistranslations.