Non-canonical Motif Research Articles

Intracellular Logistics of BST-2/Tetherin

Bone marrow stromal antigen 2 (BST-2) is a type II membrane protein with two targeting signals, one of which is located in the cytoplasmic domain and contains a non-canonical dual tyrosine-based motif responsible for its endocytosis from the plasma membrane, and the other is a C-terminal glycosylphosphatidylinositol anchor that facilitates its association with detergent-resistant membranes/lipid rafts and targeting to the apical domain in polarized epithelial cells. Due to its unusual topology at the membrane, BST-2 takes unique and complicated trafficking routes in cells. Recently, a physiological role for BST-2 as the "tetherin" molecule for viruses, especially for HIV-1, has been extensively examined. These studies have shown that the biosynthesis, intracellular trafficking, localization, and structure of human BST-2 are closely related to its antiviral activity. This review provides an overview of the intracellular logistics of human BST-2.

Transient Structure and SH3 Interaction Sites in an Intrinsically Disordered Fragment of the Hepatitis C Virus Protein NS5A

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

XPF-ERCC1: On the Bubble

In this issue of Structure, Das et al. report the structure of the helix-hairpin-helix dimerization domain of XPF bound to ssDNA. These results provide insight into the architecture of nucleotide excision repair machinery and how it interacts with damaged DNA substrates.

MED1 Coactivation of Androgen Receptor‐dependent Transcription is Mediated Through a Newly Discovered Noncanonical Binding Motif

The androgen receptor (AR) is uncommon among Nuclear receptor (NR) because of its intramolecular interaction(termed the N/C interaction) and thus coactivators can alternatively bind to surfaces in the AR amino terminal. The Mediator complex plays a key coregulatory role in steroid hormone dependent transcription and is chiefly targeted to NRs via the LxxLL‐containing MED1 subunit. However, the mechanisms by which AR recruits MED1 have remained unclear. Here we show that MED1 binds to a distinct AR amino‐terminal region termed transactivation unit‐1 (Tau‐1) via two newly discovered noncanonical α‐helical motifs of MED1. The two MED1 LxxLL motifs are not required for AR binding whereas loss of the intramolecular AR N/C interaction decreases MED1 binding. We further demonstrate that mitogen‐activated protein kinase phosphorylation of MED1 enhances the AR‐MED1 interaction in prostate cancer cells. In sum, our findings reveal a novel AR‐coactivator binding mechanism that may have clinical implications for AR activity in prostate cancer.

The Structure of the XPF-ssDNA Complex Underscores the Distinct Roles of the XPF and ERCC1 Helix- Hairpin-Helix Domains in ss/ds DNA Recognition

Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.

Effects of Non-Nearest Neighbors on the Thermodynamic Stability of RNA GNRA Hairpin Tetraloops

Currently, several models for predicting the secondary structure of RNA exist, one of which is free energy minimization using the Nearest Neighbor Model. This model predicts the lowest-free energy secondary structure from a primary sequence by summing the free energy contributions of the Watson-Crick nearest neighbor base pair combinations and any noncanonical secondary structure motif. The Nearest Neighbor Model also assumes that the free energy of the secondary structure motif is dependent solely on the identities of the nucleotides within the motif and the motif's nearest neighbors. To test the current assumption of the Nearest Neighbor Model that the non-nearest neighbors do not affect the stability of the motif, we optically melted different stem-loop oligonucleotides to experimentally determine their thermodynamic parameters. In each of these oligonucleotides, the hairpin loop sequence and the adjacent base pairs were held constant, while the first or second non-nearest neighbors were varied. The experimental results show that the thermodynamic contributions of the hairpin loop were dependent upon the identity of the first non-nearest neighbor, while the second non-nearest neighbor had a less obvious effect. These results were then used to create an updated model for predicting the thermodynamic contributions of a hairpin loop to the overall stability of the stem-loop structure.

Classification and Identification of Non-canonical Base Pairs and Structural Motifs.

The 3D structures of many ribonucleic acid (RNA) loops are characterized by highly organized networks of non-canonical interactions. Multiple computational methods have been developed to annotate structures with those interactions or automatically identify recurrent interaction networks. By contrast, the reverse problem that aims to retrieve the geometry of a look from its sequence or ensemble of interactions remains much less explored. In this chapter, we will describe how to retrieve and build families of conserved structural motifs using their underlying network of non-canonical interactions. Then, we will show how to assign sequence alignments to those families and use the software BayesPairing to build statistical models of structural motifs with their associated sequence alignments. From this model, we will apply BayesPairing to identify in new sequences regions where those loop geometries can occur.

Effects of sequence variation on differential allelic transcription factor occupancy and gene expression.

A complex interplay between transcription factors (TFs) and the genome regulates transcription. However, connecting variation in genome sequence with variation in TF binding and gene expression is challenging due to environmental differences between individuals and cell types. To address this problem, we measured genome-wide differential allelic occupancy of 24 TFs and EP300 in a human lymphoblastoid cell line GM12878. Overall, 5% of human TF binding sites have an allelic imbalance in occupancy. At many sites, TFs clustered in TF-binding hubs on the same homolog in especially open chromatin. While genetic variation in core TF binding motifs generally resulted in large allelic differences in TF occupancy, most allelic differences in occupancy were subtle and associated with disruption of weak or noncanonical motifs. We also measured genome-wide differential allelic expression of genes with and without heterozygous exonic variants in the same cells. We found that genes with differential allelic expression were overall less expressed both in GM12878 cells and in unrelated human cell lines. Comparing TF occupancy with expression, we found strong association between allelic occupancy and expression within 100 bp of transcription start sites (TSSs), and weak association up to 100 kb from TSSs. Sites of differential allelic occupancy were significantly enriched for variants associated with disease, particularly autoimmune disease, suggesting that allelic differences in TF occupancy give functional insights into intergenic variants associated with disease. Our results have the potential to increase the power and interpretability of association studies by targeting functional intergenic variants in addition to protein coding sequences.

Regulation of Androgen Receptor-dependent Transcription by Coactivator MED1 Is Mediated through a Newly Discovered Noncanonical Binding Motif

Nuclear receptor (NR) activation by cognate ligand generally involves allosteric realignment of C-terminal α-helices thus generating a binding surface for coactivators containing canonical LXXLL α-helical motifs. The androgen receptor (AR) is uncommon among NRs in that ligand triggers an intramolecular interaction between its N- and C-terminal domains (termed the N/C interaction) and that coactivators can alternatively bind to surfaces in the AR N-terminal or hinge regions. The evolutionary conserved Mediator complex plays a key coregulatory role in steroid hormone-dependent transcription and is chiefly targeted to NRs via the LXXLL-containing MED1 subunit. Whereas MED1 has been demonstrated to serve as a key transcriptional coactivator for AR, the mechanisms by which AR recruits MED1 have remained unclear. Here we show that MED1 binds to a distinct AR N-terminal region termed transactivation unit-1 (Tau-1) via two newly discovered noncanonical α-helical motifs located between MED1 residues 505 and 537. Neither of the two MED1 LXXLL motifs is required for AR binding, whereas loss of the intramolecular AR N/C interaction decreases MED1 binding. We further demonstrate that mitogen-activated protein kinase phosphorylation of MED1 enhances the AR-MED1 interaction in prostate cancer cells. In sum, our findings reveal a novel AR-coactivator binding mechanism that may have clinical implications for AR activity in prostate cancer.

An enumerative stepwise ansatz enables atomic-accuracy RNA loop modeling

Atomic-accuracy structure prediction of macromolecules should be achievable by optimizing a physically realistic energy function but is presently precluded by incomplete sampling of a biopolymer's many degrees of freedom. We present herein a working hypothesis, called the "stepwise ansatz," for recursively constructing well-packed atomic-detail models in small steps, enumerating several million conformations for each monomer, and covering all build-up paths. By making use of high-performance computing and the Rosetta framework, we provide first tests of this hypothesis on a benchmark of 15 RNA loop-modeling problems drawn from riboswitches, ribozymes, and the ribosome, including 10 cases that are not solvable by current knowledge-based modeling approaches. For each loop problem, this deterministic stepwise assembly method either reaches atomic accuracy or exposes flaws in Rosetta's all-atom energy function, indicating the resolution of the conformational sampling bottleneck. As a further rigorous test, we have carried out a blind all-atom prediction for a noncanonical RNA motif, the C7.2 tetraloop/receptor, and validated this model through nucleotide-resolution chemical mapping experiments. Stepwise assembly is an enumerative, ab initio build-up method that systematically outperforms existing Monte Carlo and knowledge-based methods for 3D structure prediction.

Inhibitory Member of the Apoptosis-stimulating Proteins of the p53 Family (iASPP) Interacts with Protein Phosphatase 1 via a Noncanonical Binding Motif

Although kinase mutations have been identified in various human diseases, much less is known about protein phosphatases. Here, we show that all apoptosis-stimulating proteins of p53 (ASPP) family members can bind protein phosphatase 1 (PP1) via two distinct interacting motifs. ASPP2 interacts with PP1 through an RVXF PP1 binding motif, whereas the inhibitory member of the ASPP family (iASPP) interacts with PP1 via a noncanonical motif (RNYF) that is located within its Src homology 3 domain (SH3). Phe-815 is crucial in mediating iASPP/PP1 interaction, and iASPP(F815A) fails to inhibit the transcriptional and apoptotic function of p53. This study identifies iASPP as a new binding partner of PP1, interacting through a noncanonical PP1 binding motif.

Evolutionary Remodeling of βγ-Crystallins for Domain Stability at Cost of Ca2+ Binding

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.

A Comprehensive View of Nuclear Receptor Cancer Cistromes

Nuclear receptors comprise a superfamily of ligand-activated transcription factors that play important roles in both physiology and diseases including cancer. The technologies of chromatin immunoprecipitation followed by array hybridization (ChIP-chip) or massively parallel sequencing (ChIP-seq) has been used to map, at an unprecedented rate, the in vivo genome-wide binding (cistrome) of nuclear receptors in both normal and cancer cells. We developed a curated database of 88 nuclear receptor cistrome data sets and other associated high-throughput data sets including 121 collaborating factor cistromes, 94 epigenomes, and 319 transcriptomes. Through integrative analysis of the curated nuclear receptor ChIP-chip/seq data sets, we discovered novel factor-specific noncanonical motifs that may have important regulatory roles. We also revealed a common feature of nuclear receptor pioneering factors to recognize relatively short and AT-rich motifs. Most nuclear receptors bind predominantly to introns and distal intergenetic regions, and binding sites closer to transcription start sites were found to be neither stronger nor more evolutionarily conserved. Interestingly, while most nuclear receptors appear to be predominantly transcriptional activators, our analysis suggests that the binding of ESR1, RARA, and RARG has both activating and repressive effects. Through meta-analysis of different omic data of the same cancer cell line model from multiple studies, we generated consensus cistrome and expression profiles. We further made probabilistic predictions of the nuclear receptor target genes by integrating cistrome and transcriptome data and validated the predictions using expression data from tumor samples. The final database, with comprehensive cistrome, epigenome, and transcriptome data sets and downstream analysis results, constitutes a valuable resource for the nuclear receptor and cancer community.

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.

CUP promotes deadenylation and inhibits decapping of mRNA targets

CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1-CCR4-NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs.

Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism

Apoptosis is a natural process during animal development for the programmed removal of superfluous cells. During apoptosis general protein synthesis is reduced, but the synthesis of cell death proteins is enhanced. Selective translation has been attributed to modification of the protein synthesis machinery to disrupt cap-dependent mRNA translation and induce a cap-independent mechanism. We have previously shown that disruption of the balance between cap-dependent and cap-independent C. elegans eIF4G isoforms (IFG-1 p170 and p130) by RNA interference promotes apoptosis in developing oocytes. Germ cell apoptosis was accompanied by the appearance of the Apaf-1 homolog, CED-4. Here we show that IFG-1 p170 is a native substrate of the worm executioner caspase, CED-3, just as mammalian eIF4GI is cleaved by caspase-3. Loss of Bcl-2 function (ced-9ts) in worms induced p170 cleavage in vivo, coincident with extensive germ cell apoptosis. Truncation of IFG-1 occurred at a single site that separates the cap-binding and ribosome-associated domains. Site-directed mutagenesis indicated that CED-3 processes IFG-1 at a non-canonical motif, TTTD456. Coincidentally, the recognition site was located 65 amino acids downstream of the newly mapped IFG-1 p130 start site suggesting that both forms support cap-independent initiation. Genetic evidence confirmed that apoptosis induced by loss of ifg-1 p170 mRNA was caspase (ced-3) and apoptosome (ced-4/Apaf-1) dependent. These findings support a new paradigm in which modal changes in protein synthesis act as a physiological signal to initiate cell death, rather than occur merely as downstream consequences of the apoptotic event.

Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7

The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism. However, the actual cleavage site in the splicing process was not determined for 13 interrupted tRNAs because of the presence of the same nucleotides at both 5′ and 3′ border regions of each intron. The cleavage sites for all the introns, which were determined by an in vitro cleavage experiment with recombinant splicing endonuclease as well as cDNA sequencing of the spliced tRNAs, indicated that non-canonical BHB structure motifs were also recognized and processed by the splicing machinery in this organism. This is the first report to empirically determine the actual cleavage and splice sites of introns in the whole set of archaeal tRNA genes, and reassigns the exon–intron borders with a novel and more plausible non-canonical BHB structure.

The munc13-4–rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane

AbstractCytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4–rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4–rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.

RNA targeting gets competitive

![Figure][1] Compared to control neurons (left), BC1 RNA (white) isn’t delivered to dendrites in cells lacking hnRNP A2 (right). Muslimov et al. reveal how noncanonical structural motifs target RNAs to neuronal dendrites and describe how this might go awry in a neurodegenerative disease

A Conserved Non-canonical Motif in the Pseudoactive Site of the ROP5 Pseudokinase Domain Mediates Its Effect on Toxoplasma Virulence

The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATP-binding motifs for non-catalytic signaling through allostery.