The structure and catalytic mechanism of a pseudoknot-containing hammerhead ribozyme.

We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozyme that closely resembles the pistol ribozyme, with essentially identical secondary structure and connectivity. The activity is more sensitive to deletion of the G8 2'OH than to the absence of magnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead, and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line, and the G8 2'OH is well positioned to act as general acid, being directed towards the O5' leaving group, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, the local structure close to the cleavage site differs, and the PK hammerhead retains its unique mechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes under standard conditions.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1.

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.

Catalytic mechanism and pH dependence of a methyltransferase ribozyme (MTR1) from computational enzymology.

A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4 kcal·mol-1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools.

Natural Antibodies Mediate Protection Against Acinetobacter baumannii Respiratory Infections.

BACKGROUND: Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. METHODS: This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell-deficient (Rag2-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. RESULTS: Our results showed that intranasally infected Rag2-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. CONCLUSIONS: Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis.

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Structure and mechanism of a methyltransferase ribozyme.

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

Validation and deployment of a direct saliva real-time RT-PCR test on pooled samples for COVID-19 surveillance testing.

A direct, real-time reverse transcriptase PCR test on pooled saliva was validated in 2,786 participants against oropharyngeal swabs. Among asymptomatic/pre-symptomatic participants, the test was found to be in 99.21% agreement and 45% more sensitive than contemporaneous oropharyngeal swabs. The test was then used for surveillance testing on 44,242 saliva samples from asymptomatic participants. Those whose saliva showed evidence of SARS-CoV-2 within 50 cycles of amplification were referred for confirmatory testing, with 87% of those tested by nasal swab within 72 hours receiving a positive diagnostic result on Abbott ID NOW or real-time PCR platforms. Median Ct values on the saliva PCR for those with a positive and negative confirmatory tests was 30.67 and 35.92 respectively, however, binary logistic regression analysis of the saliva Ct values indicates that Ct thresholds as high as 47 may be useful in a surveillance setting. Overall, data indicate that direct RT-PCR testing of pooled saliva samples is an effective method of SARS-CoV-2 surveillance.

FCRL1 Regulates B Cell Receptor-Induced ERK Activation through GRB2.

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1 -/- mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.

A simplified method for separating renal MPCs using SLAMF9.

Mononuclear phagocytes comprise an array of tissue-resident and monocyte-derived cells with important roles in tissue homeostasis and resistance to infection. Their diverse phenotypes make functional characterization within tissues challenging, because multiple surface markers are typically required for subset identification and isolation by cell sorting methods. Analysis of SLAMF9 expression within renal mononuclear phagocyte populations by multi-parametric flow cytometry indicates that SLAMF9 is a specific marker for identification of kidney-resident CD45+ CD11c+ MHC-II+ cells corresponding to prominent tissue-resident MPC populations derived from dendritic cell progenitors in adult mice. High SLAMF9 expression was sufficient to identify and sort these cells from disaggregated tissue using a user-operated cell sorter. The population can be further subdivided according to expression of CD11b and CD14 to identify IRF8high cDC1 cells and cleanly separate the CD11bhigh F4/80low and CD11bint F4/80high CD11c+ MPC subsets. Therefore, SLAMF9 expression allows for the identification and sorting of kidney-resident CD11b+ CD11c+ CD64+ F4/80+ CX3 CR1+ MHC-II+ MPCs without the need for complex antibody panels or reporter mice, simplifying isolation of these cells for study ex vivo.

Structure and folding of four putative kink turns identified in structured RNA species in a test of structural prediction rules.

k-Turns are widespread key architectural elements that occur in many classes of RNA molecules. We have shown previously that their folding properties (whether or not they fold into their tightly kinked structure on addition of metal ions) and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab (1). We have analyzed the ion-dependence of folding of the four k-turns using fluorescence resonance energy transfer, and determined the conformation of two of them using X-ray crystallography. We find that the experimental data fully conform to both the predicted folding and conformational properties. We conclude that our folding rules are robust, and can be applied to new k-turns of unknown characteristics with confidence.

The potential versatility of RNA catalysis.

It is commonly thought that in the early development of life on this planet RNA would have acted both as a store of genetic information and as a catalyst. While a number of RNA enzymes are known in contemporary cells, they are largely confined to phosphoryl transfer reactions, whereas an RNA based metabolism would have required a much greater chemical diversity of catalysis. Here we discuss how RNA might catalyze a wider variety of chemistries, and particularly how information gleaned from riboswitches could suggest how ribozymes might recruit coenzymes to expand their chemical range. We ask how we might seek such activities in modern biology. This article is categorized under: RNA-Based Catalysis > Miscellaneous RNA-Catalyzed Reactions Regulatory RNAs/RNAi/Riboswitches > Riboswitches RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.

Evidence for the loss and recovery of SLAMF9 during human evolution: implications on Dollo's law.

Signaling lymphocyte activation molecule family member 9 (SLAMF9) is a cell surface protein of the CD2/SLAM family of leukocyte surface receptors. It is conserved throughout mammals and has roles in the initiation of inflammatory responses and regulation of plasmacytoid dendritic cell function. Through comparison of reference sequences encoding SLAMF9 in human, mouse, and primate sequences, we have determined that the SLAMF9 gene underwent successive mutation events, resulting in the loss of the protein and subsequent recovery of a less stable version. The mutations included a single base pair deletion in the second exon and a change in the splice acceptor site of that same exon. These changes would have had the effect of creating and later repairing a frameshift in the coding sequence. These events took place since the divergence of the human lineage from the chimpanzee-human last common ancestor and represent the first known case of the functional loss and recovery of a gene within the human lineage.

Early Assessment of Molecular Progression and Response by Whole-genome Circulating Tumor DNA in Advanced Solid Tumors.

Treatment response assessment for patients with advanced solid tumors is complex and existing methods require greater precision. Current guidelines rely on imaging, which has known limitations, including the time required to show a deterministic change in target lesions. Serial changes in whole-genome (WG) circulating tumor DNA (ctDNA) were used to assess response or resistance to treatment early in the treatment course. Ninety-six patients with advanced cancer were prospectively enrolled (91 analyzed and 5 excluded), and blood was collected before and after initiation of a new, systemic treatment. Plasma cell-free DNA libraries were prepared for either WG or WG bisulfite sequencing. Longitudinal changes in the fraction of ctDNA were quantified to retrospectively identify molecular progression (MP) or major molecular response (MMR). Study endpoints were concordance with first follow-up imaging (FFUI) and stratification of progression-free survival (PFS) and overall survival (OS). Patients with MP (n = 13) had significantly shorter PFS (median 62 days vs. 310 days) and OS (255 days vs. not reached). Sensitivity for MP to identify clinical progression was 54% and specificity was 100%. MP calls were from samples taken a median of 28 days into treatment and 39 days before FFUI. Patients with MMR (n = 27) had significantly longer PFS and OS compared with those with neither call (n = 51). These results demonstrated that ctDNA changes early after treatment initiation inform response to treatment and correlate with long-term clinical outcomes. Once validated, molecular response assessment can enable early treatment change minimizing side effects and costs associated with additional cycles of ineffective treatment.

Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen-presenting cells and promotes resistance to Salmonella infection.

Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen-presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b+ , Ly6C- , CD11clow , F4/80low , MHC-II+ , CX3 CR1+ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80low ), but not large (F4/80high ), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9-/- mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up-regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte-macrophage colony-stimulating factor, tumour necrosis factor-α, and interleukin-1ß. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.

Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2' nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2'-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2' of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

Structure-guided design of a high-affinity ligand for a riboswitch.

We have designed structure-based ligands for the guanidine-II riboswitch that bind with enhanced affinity, exploiting the twin binding sites created by loop-loop interaction. We synthesized diguanidine species, comprising two guanidino groups covalently connected by Cn linkers where n = 4 or 5. Calorimetric and fluorescent analysis shows that these ligands bind with a 10-fold higher affinity to the riboswitch compared to guanidine. We determined X-ray crystal structures of the riboswitch bound to the new ligands, showing that the guanidino groups are bound to both nucleobases and backbone within the binding pockets, analogously to guanidine binding. The connecting chain passes through side openings in the binding pocket and traverses the minor groove of the RNA. The combination of the riboswitch loop-loop interaction and our novel ligands has potential applications in chemical biology.