1HN, 13C, and 15N backbone resonance assignments of the SET/TAF-1ß/I2PP2A oncoprotein (residues 23-225).

SET (TAF-1ß/I2PP2A) is a ubiquitously expressed, multifunctional protein that plays a role in regulating diverse cellular processes, including cell cycle progression, migration, apoptosis, transcription, and DNA repair. SET expression is ubiquitous across all cell types. However, it is overexpressed or post-translationally modified in several solid tumors and blood cancers, where expression levels are correlated with worsening clinical outcomes. SET exerts its oncogenic effects primarily through the formation of antagonistic protein complexes with the tumor suppressor, protein phosphatase 2A (PP2A), and the well-known metastasis suppressor, nm23-H1. PP2A inhibition is often observed as a secondary driver of tumorigenesis and metastasis in human cancers. Preclinical studies have shown that the pharmacological reactivation of PP2A combined with potent inhibitors of the primary driver oncogene produces synergistic cell death and decreased drug resistance. Therefore, the development of novel inhibitors of the SET-PP2A interaction presents an attractive approach to reactivation of PP2A, and thereby, tumor suppression. NMR provides a unique platform to investigate protein targets in their natively folded state to identify protein and small-molecule ligands and report on the protein internal dynamics. The backbone 1HN, 13C, and 15N NMR resonance assignments were completed for the 204 amino acid nucleosome assembly protein-1 (NAP-1) domain of the human SET oncoprotein (residues 23-225). These assignments provide a vital first step toward the development of novel PP2A reactivators via SET-selective inhibition.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress.

How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.

1HN, 13C, and 15N backbone resonance assignments of the human DNA ligase 3 DNA-binding domain (residues 257-477).

In mammalian cells, the process of DNA ligation is necessary during DNA replication to create an intact "lagging" strand from a series of smaller Okazaki fragments and to repair DNA strand breaks that arise either due to the direct action of a DNA damaging agent or as a consequence of DNA damage excision during DNA repair. In humans, there are three genes that encode for members of the DNA ligase family (LIG1, LIG3 and LIG4) (Ellenberger and Tomkinson in Ann Rev Biochem 77:313-338. 2008). Although these genes code for polypeptides with overlapping functions in the nucleus, the only mitochondrial DNA ligase (DNA ligase IIIα), which is essential for mitochondrial genome maintenance, is encoded by the LIG3 gene (Lakshmipathy and Campbell in Mol Cell Biol 19:3869-3876, 1999; Zong et al. in Mol Cell 61:667-676, 2016) Because mitochondria play a central and multifunctional role in malignant tumor progression, there is emerging interest in targeting key mitochondrial proteins. Notably, there is evidence in pre-clinical models that inhibitors of DNA ligase IIIα, which is frequently up-regulated in cancer, preferentially target cancer cells via their effect on mitochondria (Zong et al. 2016). Since NMR spectroscopy provides unique capabilities for identifying small molecules that bind specifically to DNA ligase IIIα versus the other DNA ligases), the backbone 1HN, 13C, and 15N NMR resonance assignments were completed for a 222 amino acid DNA-binding domain of human DNA ligase III. These NMR assignments represent a vital first step towards developing DNA ligase III-selective inhibitors.

The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.

The su(var)3-9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET-FTY720 or SET-ceramide, and mechanism of FTY720-dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET-FTY720 or SET-ceramide complexes by NMR spectroscopy. FTY720-ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A-Aß, PP2A-B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET-FTY720 complex in A549 cells. Various PP2A-associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720-SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET-PP2A inhibitory complex, leading to resistance to FTY720-dependent PP2A activation.-De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.

1H, 13C, and 15N resonance assignments of N-acetylmuramyl-L-alanine amidase (AmiC) N-terminal domain (NTD) from Neisseria gonorrhoeae.

Gonorrhea infections are becoming more difficult to treat due to the prevalence of strains exhibiting resistance to antibiotics and new therapeutic approaches are needed. N-acetylmuramyl-L-alanine amidase (AmiC) from Neisseria gonorrhoeae is a hydrolase that functions during cell division by cleaving the bond between the N-acetylmuramyl and L-alanine moieties of peptidoglycan. Inhibiting this enzyme offers the prospect of restoring the efficacy of existing antibiotics as treatments against N. gonorrhoeae. Of its two domains, the C-terminal domain catalyses the hydrolysis reaction and the N-terminal domain (NTD) is believed to target AmiC to its peptidoglycan substrate. Here, we report the 1H, 13C, and 15N resonance assignments of a 131 amino acid NTD construct of AmiC by heteronuclear NMR spectroscopy. The assignments represent the first for N. gonorrhoeae AmiC-NTD, laying the groundwork for detailed examination of its structure and dynamics, and providing a platform for new drug discovery efforts to address antimicrobial-resistant N. gonorrhoeae.

Balance between senescence and apoptosis is regulated by telomere damage-induced association between p16 and caspase-3.

Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage-dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage-dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT) but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2-/- mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Furthermore, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired caspase-3 association did not prevent telomere damage-induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis. These results suggest that p16 plays a direct role in telomere damage-dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage-dependent senescence and apoptosis with regards to aging and cancer.

The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism.

The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed. Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.

Crystal structure of the human heterogeneous ribonucleoprotein A18 RNA-recognition motif.

The heterogeneous ribonucleoprotein A18 (hnRNP A18) is upregulated in hypoxic regions of various solid tumors and promotes tumor growth via the coordination of mRNA transcripts associated with pro-survival genes. Thus, hnRNP A18 represents an important therapeutic target in tumor cells. Presented here is the first X-ray crystal structure to be reported for the RNA-recognition motif of hnRNP A18. By comparing this structure with those of homologous RNA-binding proteins (i.e. hnRNP A1), three residues on one face of an antiparallel ß-sheet (Arg48, Phe50 and Phe52) and one residue in an unstructured loop (Arg41) were identified as likely to be involved in protein-nucleic acid interactions. This structure helps to serve as a foundation for biophysical studies of this RNA-binding protein and structure-based drug-design efforts for targeting hnRNP A18 in cancer, such as malignant melanoma, where hnRNP A18 levels are elevated and contribute to disease progression.

(1)H(N), (13)C, and (15)N resonance assignments of the CDTb-interacting domain (CDTaBID) from the Clostridium difficile binary toxin catalytic component (CDTa, residues 1-221).

Once considered a relatively harmless bacterium, Clostridium difficile has become a major concern for healthcare facilities, now the most commonly reported hospital-acquired pathogen. C. difficile infection (CDI) is usually contracted when the normal gut microbiome is compromised by antibiotic therapy, allowing the opportunistic pathogen to grow and produce its toxins. The severity of infection ranges from watery diarrhea and abdominal cramping to pseudomembranous colitis, sepsis, or death. The past decade has seen a marked increase in the frequency and severity of CDI among industrialized nations owing directly to the emergence of a highly virulent C. difficile strain, NAP1. Along with the large Clostridial toxins expressed by non-epidemic strains, C. difficile NAP1 produces a binary toxin, C. difficile transferase (CDT). As the name suggests, CDT is a two-component toxin comprised of an ADP-ribosyltransferase (ART) component (CDTa) and a cell-binding/translocation component (CDTb) that function to destabilize the host cytoskeleton by covalent modification of actin monomers. Central to the mechanism of binary toxin-induced pathogenicity is the formation of CDTa/CDTb complexes at the cell surface. From the perspective of CDTa, this interaction is mediated by the N-terminal domain (residues 1-215) and is spatially and functionally independent of ART activity, which is located in the C-terminal domain (residues 216-420). Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of a 221 amino acid, ~26 kDa N-terminal CDTb-interacting domain (CDTaBID) construct by heteronuclear NMR spectroscopy. These NMR assignments represent the first component coordination domain for a family of Clostridium or Bacillus species harboring ART activity. Our assignments lay the foundation for detailed solution state characterization of structure-function relationships, toxin complex formation, and NMR-based drug discovery efforts.

Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein.

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions in the p2 pocket dominated affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 19-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukemia.

Structural Re-engineering of the α-Helix Mimetic JY-1-106 into Small Molecules: Disruption of the Mcl-1-Bak-BH3 Protein-Protein Interaction with 2,6-Di-Substituted Nicotinates.

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 µm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

1H, 13C, and 15N resonance assignments of an enzymatically active domain from the catalytic component (CDTa, residues 216-420) of a binary toxin from Clostridium difficile.

Clostridium difficile is a bacterial pathogen and is the most commonly reported source of nosocomial infection in industrialized nations. Symptoms of C. difficile infection (CDI) include antibiotic-associated diarrhea, pseudomembranous colitis, sepsis and death. Over the last decade, rates and severity of hospital infections in North America and Europe have increased dramatically and correlate with the emergence of a hypervirulent strain of C. difficile characterized by the presence of a binary toxin, CDT (C. difficile toxin). The binary toxin consists of an enzymatic component (CDTa) and a cellular binding component (CDTb) that together form the active binary toxin complex. CDTa harbors a pair of structurally similar but functionally distinct domains, an N-terminal domain (residues 1-215; (1-215)CDTa) that interacts with CDTb and a C-terminal domain (residues 216-420; (216-420)CDTa) that harbors the intact ADP-ribosyltransferase (ART) active site. Reported here are the (1)H, (13)C, and (15)N backbone resonance assignments of the 23 kDa, 205 amino acid C-terminal enzymatic domain of CDTa, termed (216-420)CDTa. These NMR resonance assignments for (216-420)CDTa represent the first for a family of ART binary toxins and provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

Backbone ¹HN, ¹³C, and ¹5N resonance assignments of the tandem RNA-binding domains of human DGCR8.

Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493-720 revealed that each dsRBD adopts the canonical αßßßα fold. However, several residues located in important flexible regions including the ß1-ß2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8(core) (residues 493-706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.

Plant viral suppressors of RNA silencing.

RNA silencing is an ancient eukaryotic pathway in which double stranded RNA (dsRNA) triggers destruction of related RNAs in the cell. Early studies in plants pointed to a role for this pathway as a defense against viruses. Most known plant viruses have RNA genomes and replicate via dsRNA intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silencing targets later in infection. Because RNA silencing is an antiviral mechanism, it is not surprising that many plant viruses encode suppressors of RNA silencing. This review focuses on the currently known plant virus encoded suppressors of silencing and the functional assays used to identify these proteins. Because they interfere with silencing at different points in the pathway, these viral suppressors are powerful tools to help unravel the mechanism of RNA silencing in plants.