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Abstract: Despite the theoretical advantages of phosphorus single-wavelength anomalous diffraction (P-SAD) for nucleic acid phasing, its application remains limited due to high atomic displacement parameters and an unfavourable ratio of unique reflections to anomalous scatterers. In this study, we report the crystal structure of an RNA complex composed of four strands, which was solved by experimental phasing after AlphaFold3 failed to produce reliable models. Bromine single-wavelength anomalous diffraction (Br-SAD) data were collected at 0.916 Å on beamline I04 at Diamond Light Source, while phosphorus anomalous data were obtained at 3.024 Å on beamline I23. The structure was successfully phased using bromine anomalous scattering, and phosphorus anomalous peaks corroborated the backbone positions and validated the model. Attempts to phase the structure directly from phosphorus data failed, consistent with theoretical predictions that successful SAD phasing requires a significantly higher reflection-to-scatterer ratio. The final models reveal an RNA complex stabilized by Watson–Crick and Hoogsteen base pairing, forming a pseudo-helical complex instead of the anticipated hairpin stem-loop, likely reflecting crystallization artefacts. This work demonstrates the complementary use of bromine and phosphorus anomalous signals in RNA crystallography.

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Abstract: Angiotensin I-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase involved in blood pressure regulation through proteolysis of angiotensin I (Ang-I) into the potent vasoconstrictor, angiotensin II (Ang-II). Inhibition of ACE is therefore used for the treatment of hypertension, heart failure, myocardial infarction, stroke and chronic kidney disease. Current ACE inhibitors (ACEi) bind both the N- and C-catalytic domains of ACE (referred to as nACE and cACE), and this has been linked to the occurrence of side effects due to the wide substrate specificity of ACE. The development of domain selective ACEi with reduced side effects is therefore key for improved therapeutic intervention. Understanding how current ACEi bind nACE and cACE, and their differences in domain selectivity should aid structure-based development of more selective ACEi by identifying different chemical groups that increase or decrease selectivity. We present the kinetic and structural characterisation of nACE and cACE with three thiolate ACEi, captopril (Ki, nACE = 2.53 nm and cACE = 2.04 nm), rentiapril (monomer Ki, nACE = 2.22 nm and cACE = 6.77 nm) and zofenoprilat (Ki, nACE = 2.86 nm and cACE = 0.61 nm). Detailed structural analysis indicated that the S2′ subsite likely contributes to the variation in domain selectivity observed for rentiapril and zofenoprilat due to differences in hydrophobicity and displacement of water molecules that contribute to ACE's hydration shell. Interestingly, in the cACE crystal structure, rentiapril bound as a dimer, and kinetic data revealed that both the monomeric and dimeric (dimer Ki, nACE = 15.11 nm and cACE = 36.38 nm) forms of rentiapril inhibit ACE with nanomolar affinity.

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Abstract: Lytic polysaccharide monooxygenases ([L]PMOs) are copper-containing enzymes that catalyse cleavage of the glycosidic bond, a process central to microbial biomass degradation. Here, we describe electrochemical methods used to investigate the Cu2+/1+ redox chemistry and the polysaccharide-free catalytic activity of two AA10 LPMOs: CjAA10B from Cellvibrio japonicus and CfAA10 from Cellulomonas fimi. Immobilisation of these enzymes on the surface of a graphite electrode allows for direct electrochemical measurements of Cu2+/1+ redox cycling as well as the ability of both LPMOs to reduce H2O2 vs O2. These measurements can be advantageous when compared to biological dye assays as they provide direct kinetic measurements and allow for investigation over a wider range of environmental conditions. Values of kcat and KM- are reported for H2O2 and O2 reduction by CjAA10B and CfAA10 from pH 5–7, with CfAA10 consistently outperforming CjAA10B. Both enzymes perform faster catalysis with H2O2 but when comparing the affinity-coupled specificity constant (kcat/KM), the LPMOs perform similarly with both H2O2 and O2, suggesting both substrates are viable. We also note an increase in redox signals as pH is decreased that correlates with EPR data suggesting a second species is formed <pH 5, postulated to occur due to the protonation of a glutamate residue (pKa ∼ 4.6). The increase in signal size with decreasing pH that is seen for the non-catalytic Cu2+/1+ transition is interpreted in light of an increasing proportion of electroactive species at low pH; such a change in activity with pH is notably not observed in the presence of substrate (H2O2 or O2). This suggests that substrate binding modulates the active site, disrupting the effect of protonation. These findings establish electrochemistry as a powerful tool for probing LPMO activity.

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Abstract: Most prokaryotes divide using filaments of the tubulin-like FtsZ protein, while some archaea employ instead ESCRT-III-like proteins and their filaments for cell division and cytokinesis. The alternative archaeal system comprises Cdv proteins and is thought to bear some resemblance to ESCRT-III-based membrane remodeling in other domains of life, including eukaryotes, especially during abscission. Here, we present biochemical, crystallographic, and cryo-EM studies of the Sulfolobus Cdv machinery. CdvA, an early non-ESCRT component, adopts a PRC‐domain/coiled-coil fold and polymerizes into long double-stranded helical filaments, mainly via hydrophobic interfaces. Monomeric CdvB adopts the canonical ESCRT-III fold in both a closed and a distinct “semiopen” conformation. Soluble CdvB2 filaments are composed of subunits in the closed state, appearing to transition to the open, active state only when polymerized on membranes. Short N-terminal amphipathic helices in all CdvB paralogues, B, B1, and B2, mediate membrane binding and are required for liposome recruitment in vitro. We provide a molecular overview of archaeal ESCRT-III-based cytokinesis machinery, the definitive demonstration that CdvB proteins are bona fide ESCRT-III homologues, and reveal the molecular basis for membrane engagement. Thus, we illuminate conserved principles of ESCRT-mediated membrane remodeling and extend them to an anciently diverged archaeal lineage.

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Abstract: SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.