Activation of the <i>glmS</i> Ribozyme Nucleophile via Overdetermined Hydrogen Bonding

RNA enzymes, or ribozymes, catalyze internal phosphodiester bond cleavage using diverse catalytic strategies. These include the four classic strategies: in-line nucleophilic attack, deprotonation of the 2′-OH nucleophile, protonation of the 5′-O leaving group, and stabilization of developing charge on the nonbridging oxygen atoms of the scissile phosphate. In addition, we recently identified two additional ribozyme strategies: acidification of the 2′-OH and release of the 2′-OH from inhibitory interactions. Herein, we report inverse thio effects in the presence of <i>glmS</i> ribozyme variants and a 1-deoxyglucosamine 6-phosphate cofactor analogue and demonstrate that activation of the 2′-OH nucleophile is promoted by competitive hydrogen bonding among diverse ribozyme moieties for the <i>pro</i>-<i>R</i>_P nonbridging oxygen. We conclude that the <i>glmS</i> ribozyme uses an overdetermined set of competing hydrogen bond donors in its active site to ensure potent activation and regulation by the cofactor. Nucleophile activation through competitive, overdetermined hydrogen bonding could be a general strategy for ribozyme activation and may be applicable for controlling the function of ribozymes and riboswitches in the laboratory

Role of the Active Site Guanine in the <i>glmS</i> Ribozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

The <i>glmS</i> ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and p<i>K</i>_a calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40­(N1) or possibly A-1­(O2′), which would be followed by proton transfer from G40­(N1) to A-1­(O2′). After this initial deprotonation, A-1­(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40­(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1­(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1­(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg²⁺ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated p<i>K</i>_a rather than a p<i>K</i>_a shifted toward neutrality, although a balance among the p<i>K</i>_a’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40

Role of the Active Site Guanine in the <i>glmS</i> Ribozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

The <i>glmS</i> ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and p<i>K</i>_a calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40­(N1) or possibly A-1­(O2′), which would be followed by proton transfer from G40­(N1) to A-1­(O2′). After this initial deprotonation, A-1­(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40­(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1­(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1­(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg²⁺ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated p<i>K</i>_a rather than a p<i>K</i>_a shifted toward neutrality, although a balance among the p<i>K</i>_a’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40

Assessing the Potential Effects of Active Site Mg²⁺ Ions in the <i>glmS</i> Ribozyme–Cofactor Complex

Ribozymes employ diverse catalytic strategies in their self-cleavage mechanisms, including the use of divalent metal ions. This work explores the effects of Mg²⁺ ions in the active site of the <i>glmS</i> ribozyme–GlcN6P cofactor complex using computational methods. Deleterious and potentially beneficial effects of an active site Mg²⁺ ion on the self-cleavage reaction were identified. The presence of a Mg²⁺ ion near the scissile phosphate oxygen atoms at the cleavage site was determined to be deleterious, and thereby anticatalytic, due to electrostatic repulsion of the cofactor, disruption of key hydrogen-bonding interactions, and obstruction of nucleophilic attack. On the other hand, the presence of a Mg²⁺ ion at another position in the active site, the Hoogsteen face of the putative base, was found to avoid these deleterious effects and to be potentially catalytically favorable owing to the stabilization of negative charge and p<i>K</i>_a shifting of the guanine base

IL-38 blockade induces anti-tumor immunity by abrogating tumor-mediated suppression of early immune activation

ABSTRACTImmune checkpoint inhibitors that overcome T cell suppressive mechanisms in tumors have revolutionized the treatment of cancer but are only efficacious in a small subset of patients. Targeting suppressive mechanisms acting on innate immune cells could significantly improve the incidence of clinical response by facilitating a multi-lineage response against the tumor involving both adaptive and innate immune systems. Here, we show that intra-tumoral interleukin (IL)-38 expression is a feature of a large frequency of head and neck, lung and cervical squamous cancers and correlates with reduced immune cell numbers. We generated IMM20324, an antibody that binds human and mouse IL-38 proteins and inhibits the binding of IL-38 to its putative receptors, interleukin 1 receptor accessory protein-like 1 (IL1RAPL) and IL-36R. In vivo, IMM20324 demonstrated a good safety profile, delayed tumor growth in a subset of mice in an EMT6 syngeneic model of breast cancer, and significantly inhibited tumor expansion in a B16.F10 melanoma model. Notably, IMM20324 treatment resulted in the prevention of tumor growth following re-implantation of tumor cells, indicating the induction of immunological memory. Furthermore, exposure of IMM20324 correlated with decreased tumor volume and increased levels of intra-tumoral chemokines. Together, our data suggest that IL-38 is expressed in a high frequency of cancer patients and allows tumor cells to suppress anti-tumor immunity. Blockade of IL-38 activity using IMM20324 can re-activate immunostimulatory mechanisms in the tumor microenvironment leading to immune infiltration, the generation of tumor-specific memory and abrogation of tumor growth

Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2

Time-Resolved Vibrational Spectroscopy of [FeFe]-Hydrogenase Model Compounds

Model compounds have been found to structurally mimic the catalytic hydrogen-producing active site of Fe–Fe hydrogenases and are being explored as functional models. The time-dependent behavior of Fe₂(μ-S₂C₃H₆)­(CO)₆ and Fe₂(μ-S₂C₂H₄)­(CO)₆ is reviewed and new ultrafast UV- and visible-excitation/IR-probe measurements of the carbonyl stretching region are presented. Ground-state and excited-state electronic and vibrational properties of Fe₂(μ-S₂C₃H₆)­(CO)₆ were studied with density functional theory (DFT) calculations. For Fe₂(μ-S₂C₃H₆)­(CO)₆ excited with 266 nm, long-lived signals (τ = 3.7 ± 0.26 μs) are assigned to loss of a CO ligand. For 355 and 532 nm excitation, short-lived (τ = 150 ± 17 ps) bands are observed in addition to CO-loss product. Short-lived transient absorption intensities are smaller for 355 nm and much larger for 532 nm excitation and are assigned to a short-lived photoproduct resulting from excited electronic state structural reorganization of the Fe–Fe bond. Because these molecules are tethered by bridging disulfur ligands, this extended di-iron bond relaxes during the excited state decay. Interestingly, and perhaps fortuitously, the time-dependent DFT-optimized exited-state geometry of Fe₂(μ-S₂C₃H₆)­(CO)₆ with a semibridging CO is reminiscent of the geometry of the Fe₂S₂ subcluster of the active site observed in Fe–Fe hydrogenase X-ray crystal structures. We suggest these wavelength-dependent excitation dynamics could significantly alter potential mechanisms for light-driven catalysis