Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase.

CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.

Structural Transition and Antibody Binding of EBOV GP and ZIKV E Proteins from Pre-Fusion to Fusion-Initiation State.

Membrane fusion proteins are responsible for viral entry into host cells—a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion-initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion-initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola virus glycoprotein (EBOV GP) and Zika virus envelope protein (ZIKV E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion-initiation states have been described. While the fusion-initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion-initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

Identification and Characterization of a Class of MALAT1-like Genomic Loci.

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript (MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant long noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Thus, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

Optimal aggregation of FcεRI with a structurally defined trivalent ligand overrides negative regulation driven by phosphatases.

To investigate why responses of mast cells to antigen-induced IgE receptor (FcεRI) aggregation depend nonlinearly on antigen dose, we characterized a new artificial ligand, DF3, through complementary modeling and experimentation. This ligand is a stable trimer of peptides derived from bacteriophage T4 fibritin, each conjugated to a hapten (DNP). We found low and high doses of DF3 at which degranulation of mast cells sensitized with DNP-specific IgE is minimal, but ligand-induced receptor aggregation is comparable to aggregation at an intermediate dose, optimal for degranulation. This finding makes DF3 an ideal reagent for studying the balance of negative and positive signaling in the FcεRI pathway. We find that the lipid phosphatase SHIP and the protein tyrosine phosphatase SHP-1 negatively regulate mast cell degranulation over all doses considered. In contrast, SHP-2 promotes degranulation. With high DF3 doses, relatively rapid recruitment of SHIP to the plasma membrane may explain the reduced degranulation response. Our results demonstrate that optimal secretory responses of mast cells depend on the formation of receptor aggregates that promote sufficient positive signaling by Syk to override phosphatase-mediated negative regulatory signals.

Rise of the RNA machines: exploring the structure of long non-coding RNAs.

Novel, profound and unexpected roles of long non-coding RNAs (lncRNAs) are emerging in critical aspects of gene regulation. Thousands of lncRNAs have been recently discovered in a wide range of mammalian systems, related to development, epigenetics, cancer, brain function and hereditary disease. The structural biology of these lncRNAs presents a brave new RNA world, which may contain a diverse zoo of new architectures and mechanisms. While structural studies of lncRNAs are in their infancy, we describe existing structural data for lncRNAs, as well as crystallographic studies of other RNA machines and their implications for lncRNAs. We also discuss the importance of dynamics in RNA machine mechanism. Determining commonalities between lncRNA systems will help elucidate the evolution and mechanistic role of lncRNAs in disease, creating a structural framework necessary to pursue lncRNA-based therapeutics.

Conformational dependence of a protein kinase phosphate transfer reaction.

Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. With the protein in TC, the motions involved in reaction are small, with only P(gamma) and the catalytic proton moving >0.5 A. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site.

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics, hydration, sequence, and ionic effects.

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG]] or cis-[Pt(NH(3))(2) [d(ApG]] cross-links, and their corresponding unmodified duplexes. The platinated duplexes are less stable and unfold with lower T(M)s (and Delta G degrees s) in enthalpy-driven reactions, which indicates a loss of favorable base-pair stacking interactions. The folding thermodynamics and hydration effects for the first set of decamers containing the d(GpG) cross-link was investigated by a combination of titration calorimetry, density, and ultrasound techniques. The hydration parameters showed an uptake of structural water by the platinated duplex and a release of electrostricted water by the control duplex. Relative to the unmodified duplex, the folding of the platinated duplex at 20 degrees C yielded a positive Delta Delta G degrees term [and positive Delta Delta H-Delta(T Delta S) compensation] and a negative differential volume change. The opposite signs of the Delta Delta G degrees and Delta Delta V terms confirmed its uptake of structural water. Further, solvent-accessible surface areas calculations for a similar pair of dodecamer duplexes indicated that the modified duplex has a 503 oeA(2) higher polar and nonpolar surface area that is exposed to the solvent. Therefore, the incorporation of a platinum adduct in duplex DNA disrupts favorable base-pair stacking interactions, yielding a greater exposure of aromatic bases to the solvent, which in turn immobilizes structural water. The overall results correlate nicely with the results reported in the available structural data of nuclear magnetic resonance solution studies.

A structural model of the catalytic subunit-regulatory subunit dimeric complex of the cAMP-dependent protein kinase.

Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R(IIalpha)) and catalytic (C(alpha)) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, Delta(1-91)R(IIalpha)-C(alpha). The first step in the modeling utilized the crystal structures for the porcine C(alpha) and bovine Delta(1-90)R(Ialpha) or rat Delta(1-111)R(IIbeta), to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C(alpha) subunit and murine Delta(1-91)R(IIalpha) subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R(Ialpha) and R(IIbeta) crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R(Ialpha)-based model has 25% more hydrogen bonds than that based on R(IIbeta), with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 A(2)). The R(Ialpha)-based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.