Xianyang Fang

Structural model of an mRNA in complex with the bacterial chaperone Hfq

Significance Small noncoding RNAs optimize bacterial gene expression under stress and increase the virulence of many bacterial pathogens. The RNA-binding protein Hfq (host factor Q-beta phage) promotes base pairing between small RNAs and target mRNAs, but it is not known how Hfq brings the two RNAs together in the proper orientation. We used chemical footprinting, small-angle X-ray scattering, and molecular dynamics simulations to model the structure of Hfq bound to an mRNA in solution. The surprising result is that the mRNA wraps entirely around the Hfq protein, specifically contacting both surfaces. This destabilizes the mRNA structure around the small RNA target site, poising it to base pair with a complementary small RNA also bound to Hfq. The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. Here, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2′-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNA regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR)

Background: The copper-sensing operon repressor (CsoR) is representative of a large family of poorly understood copper sensors. Results: Geobacillus thermodenitrificans CsoR (Gt CsoR) is representative of CsoRs found in several human pathogens. Conclusion: Cu(I) binding induces a structural change as modeled by small angle x-ray scattering and NMR spectroscopy. Significance: This work provides new insights into copper-mediated conformational switching in CsoR family proteins. The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12–19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (Δ2–10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed.

Two ZnF-UBP Domains in Isopeptidase T (USP5)

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzymes hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrates unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.

Small-angle X-ray scattering: a bridge between RNA secondary structures and three-dimensional topological structures

Whereas the structures of small to medium-sized well folded RNA molecules often can be determined by either X-ray crystallography or NMR spectroscopy, obtaining structural information for large RNAs using experimental, computational, or combined approaches remains a major interest and challenge. RNA is very sensitive to small-angle X-ray scattering (SAXS) due to high electron density along phosphate-sugar backbones, whose scattering contribution dominates SAXS intensity. For this reason, SAXS is particularly useful in obtaining global RNA structural information that outlines backbone topologies and, therefore, molecular envelopes. Such information is extremely valuable in bridging the gap between the secondary structures and three-dimensional topological structures of RNA molecules, particularly those that have proven difficult to study using other structure-determination methods. Here we review published results of RNA topological structures derived from SAXS data or in combination with other experimental data, as well as details on RNA sample preparation for SAXS experiments.

A Stabilizing alpha/beta-Hydrophobic Core Greatly Contributes to Hyperthermostability of Archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

ABSTRACT Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCE Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

A mechanism of global shape-dependent recognition and phosphorylation of filamin by protein kinase A

Background: The mechanism of filamin Ser2152 phosphorylation by PKA is unclear. Results: Autoinhibitory filamin is resistant to phosphorylation despite exposed Ser2152, but ligand binding alters the filamin conformation, triggering PKA recognition. Conclusion: Filamin Ser2152 phosphorylation is conformation-dependent on ligand binding. Significance: The overall conformation of substrate, not just the exposed phosphorylation site, regulates the kinase substrate recognition in signaling. Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by ∼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser2152 site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser2152. These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.

Favorable contribution of the C-terminal residue K97 to the stability of a hyperthermophilic archaeal [P62A]Ssh10b

The role of residue K97 at the C-terminal end of archaeal [P62A]Ssh10b in the hyperthermostability of the protein is investigated using three K97-mutant variants: K97E-, K97A-, and DeltaK97-mutant [P62A]Ssh10b. The thermal- and GdmHCl-induced denaturation of the three mutant variants has been monitored by circular dichroism. The results reveal that the K97E mutation leads to a stronger destabilization effect than the K97A mutation by disturbing the electrostatic interaction of the salt-bridge D63-K97 and drawing an unfavorable charge-charge repulsive interaction into the structure. However, DeltaK97-[P62A]Ssh10b shows much lower stability than K97E- and K97A-mutant [P62A]Ssh10b. Analysis suggests that residue K97 at the C-terminal end makes the favorable contributions to the stability of hyperthermophilic [P62A]Ssh10b not only by the favorable electrostatic interactions with residues in close vicinity but also through maintaining the side chain packing of the surrounding residues in the C-terminal area of the protein.

The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III.

Double-stranded RNA (dsRNA)-specific RNase III proteins are required for RNA maturation and gene regulation. The mechanism of prokaryotic RNase IIIs has been well characterized, but how eukaryotic RNase IIIs (exemplified by Rnt1p, Drosha, and Dicer) work is less clear. Recently, we reported the crystal structure of Rnt1p in complex with RNA, revealing a double-ruler mechanism for substrate selection. Here, we present more structures of Rnt1p, either RNA free or RNA bound, featuring two major conformations of the enzyme. Using these structures with existing data, we describe the functional cycle of Rnt1p in five steps, selecting, loading, locking, cleavage, and releasing. We also describe atomic details of the two-Mg2+-ion catalytic mechanism that is applicable to all eukaryotic RNase III enzymes. Overall, our results indicate that substrate selection is achieved independent of cleavage, allowing the recognition of substrates with different structures while preserving the basic mechanism of cleavage.

Topological Structure Determination of RNA Using Small-Angle X-Ray Scattering

Knowledge of RNA three-dimensional topological structures provides important insight into the relationship between RNA structural components and function. It is often likely that near-complete sets of biochemical and biophysical data containing structural restraints are not available, but one still wants to obtain knowledge about approximate topological folding of RNA. In this regard, general methods for determining such topological structures with minimum readily available restraints are lacking. Naked RNAs are difficult to crystallize and NMR spectroscopy is generally limited to small RNA fragments. By nature, sequence determines structure and all interactions that drive folding are self-contained within sequence. Nevertheless, there is little apparent correlation between primary sequences and three-dimensional folding unless supplemented with experimental or phylogenetic data. Thus, there is an acute need for a robust high-throughput method that can rapidly determine topological structures of RNAs guided by some experimental data. We present here a novel method (RS3D) that can assimilate the RNA secondary structure information, small-angle X-ray scattering data, and any readily available tertiary contact information to determine the topological fold of RNA. Conformations are firstly sampled at glob level where each glob represents a nucleotide. Best-ranked glob models can be further refined against solvent accessibility data, if available, and then converted to explicit all-atom coordinates for refinement against SAXS data using the Xplor-NIH program. RS3D is widely applicable to a variety of RNA folding architectures currently present in the structure database. Furthermore, we demonstrate applicability and feasibility of the program to derive low-resolution topological structures of relatively large multi-domain RNAs.