Computational study of the furin cleavage domain of SARS-CoV-2: delta binds strongest of extant variants

We demonstrate that AlphaFold and AlphaFold Multimer, implemented within the ColabFold suite, can accurately predict the structures of the furin enzyme with known six residue inhibitory peptides. Noting the similarity of the peptide inhibitors to polybasic furin cleavage domain insertion region of the SARS-CoV-2, which begins at P681, we implement this approach to study the wild type furin cleavage domain for the virus and several mutants. We introduce mutations in silico for alpha, omicron, and delta variants, for several sequences which have been rarely observed, for sequences which have not yet been observed, for other coronaviruses (NL63, OC43, HUK1a, HUK1b, MERS, and 229E), and for the H5N1 flu. We show that interfacial hydrogen bonds between the furin cleavage domain and furin are a good measure of binding strength that correlate well with endpoint binding free energy estimates, and conclude that among all candidate viral sequences studied, delta is near the very top binding strength within statistical accuracy. However, the binding strength of several rare sequences match delta within statistical accuracy. We find that the furin S1 pocket is optimized for binding arginine as opposed to lysine. This residue, typically at sequence position five, contains the most hydrogen bonds to the furin, and hydrogen bond count for just this residue shows a strong positive correlation with the overall hydrogen bond count . We demonstrate that the root mean square backbone C-alpha fluctuation of the first residue in the furin cleavage domain has a strong negative correlation with the interfacial hydrogen bond count. We show by considering the variation with the number of basic residues that the maximum mean number of interfacial hydrogen bonds expected is 15.7 at 4 basic residues.

Simulation of the omicron variant of SARS-CoV-2 shows broad antibody escape, weakened ACE2 binding, and modest increase in furin binding

The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is significant reduction of antibody binding strength corresponding to escape, the omicron spike pays a cost in terms of weaker receptor binding. The furin cleavage domain is the same or weaker binding than the alpha variant, suggesting less viral load and disease intensity than the extant delta variant.

Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application

Disruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5Δ55–60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24–48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%–56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects.

Structural basis for allosteric regulation of Human Topoisomerase IIα

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6–7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme’s activities and opens perspective for the analysis of post-translational modifications.

Construction of a G-quadruplex-specific DNA endonuclease

A G-quadruplex-specific DNA endonuclease was constructed by fusing a RHAU G-quadruplex recognition domain with a Fok1 cleavage domain, providing a useful tool for detection of G-quadruplex structures.

DISCLOSING THE TRUE NATURE OF HESPERETIN’S ANTIGENOTOXICITY „IN VIVO“ WITHIN THE „DROSOPHILA MELANOGASTER“ SOMATIC CELLS THROUGH THE EXTENSIVE GENOTOXICAL AND STRUCTURE-BASED STUDIES

Previously unreported genotoxic and antigenotoxic potentials of hesperetin (Hes) were revealed by treating the Drosophila melanogaster (dm) whose DNA has been altered by means of O6-ethylguanine (dmGO6-Et) and O4-ethylthymine (dmTO4-Et) lesions appearance, caused by ethyl methanesulfonate (EMS), a proven alkylating agent and mutagen. Therefore, Hes potencies were determined by means of the comet assay on somatic cells level, where compound exerted no genotoxic effects but acted genotoxically as a Topoisomerase IIα (dmTopIIα) catalytic inhibitor by invading the Binding and Cleavage Domain and stabilizing the noncovalent dmTopIIα-plasmid DNA (dmPDNA) complex, as verified by the kinetoplast DNA (dmK-DNA) decatenation assays. Hes’s structure-based alignment caused compound’s A and C rings to occupy the area normally invaded by EMS, thus making a spatial barrier for the dmGO6-Et or dmTO4-Et lesions formation: the A ring C7-OH group formed hydrogen bonds (HBs) with either dmGO6 (dHB = 2.576 Å) or guanine’s N7 nitrogen (dmGN7, dHB = 2.737 Å), whereas the A ring C5-OH group formed an HB with dmTO4 (dHB = 3.548 Å). Furthermore, Hes likewise acted as a mixed-type competitive inhibitor of dmATPase, as verified by the catalytic, FRET, and structure-based studies where it affected the dmATPase dimerization and the hydrolysis of ATP, denying the metabolic energy for the catenation of ethylated G-dmDNA segment, the formation of dmTO4-Et-G-dmDNA phosphotyrosine intermediate (dmTO4-Et-G- dmDNA-PTyr785I), and the passage of ethylated T-dmDNA segment through the temporarily broken dmTO4-Et-G-dmDNA-PTyr785I, processes seen as comets. Conclusively, Hes may be used in anticancer therapy controlling the effects of alkylating agents.

EPCO-39. DICER1 SYNDROME WITH PRIMITIVE NEUROECTODERMAL TUMOR FROM SCREENING MUTATIONS OF DICER1 GENE

BACKGROUND DICER1 syndrome is a rare disorder of tumor predisposition. The DICER1 gene encodes the Dicer protein of the ribonuclease Ⅲ family and plays a critical role in miRNA creation. Patients with DICER1 syndrome-related cancer commonly harbor an additional somatic variant in exon24 or 25 of DICER1 involving one of the hotspots including p.E1705, p.D1709, p.G1809, p.D1810, or p.E1813. Herein, we firstly report the presence of hotspot missence mutation in a patient with primitive neuroectodermal tumor (PNET). METHODS The mutations in DIRCER1 were searched in the tumor tissue or peripheral blood from 4,349 patients with different types of cancer via next generation sequencing. Among them, comprehensive genomic profiling (CGP) with 131 CNS cancer-related genes and 4 brain tumor-related chromosome structure variations was performed in 230 cases, and CGP with 539 cancer-related genes was administrated in 4,119 cases. The histopathology diagnosis of each case was confirmed via hematoxylin and eosin staining. RESULTS A number of 99 mutations in the exon and intron regions of DICER1 gene were observed from 88 patients in 4,349 cases. The ratio of somatic variants located on exon 24 and 25 where both encoded the RNase Ⅲb cleavage domain to all mutations was 9.09% (9/99). There were 3 and 6 somatic variants in the exon 24 and 25, respectively. Among the 9 mutations, 4 of them were p.E1813, one class of hotspots located on exon 25 leading to DICER1 syndrome, and the other 4 hotspots were not observed. Among 4 cases of DICER1 syndrom, pleuropulmenary blastoma was common, rhabdomyosarcomas arisen in two cases were also reported before, and the last one was patient with PNET. CONCLUSIONS In this study, we analyze the mutation distribution of DICER1 in cancer patients, report firstly a patient with PNET harbors a hotspot, and widen and deepen the understanding of DICER1 syndrome.

Structural basis for the allosteric regulation of Human Topoisomerase 2α

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Top2α isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Top2α nucleoprotein complex in different conformations solved at subnanometer resolutions. Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme’s activities and opens perspective for the analysis of post-translational modifications.

The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain

We analyzed the SARS-CoV-2 spike (S) protein amino acid sequence extracted from 11,542 viral genomic sequences submitted to the Global Initiative on Sharing All Influenza Data (GISAID) database through April 27, 2020. Consistent with prior reports, we found a major S protein mutation, D614 to G614, that was represented in 56% of all the analyzed sequences. All other mutations combined were less than 10%. After parsing the data geographically, we found most of the Chinese patient samples showed D614 (97%). By contrast, most patient samples in many European countries showed G614 (51 to 88%). In the United States, the genotypic distribution in California and Washington was similar to Asian countries, while the distribution in other US states was comparable to Europe. We observed a dramatic increase in the frequency of G614 over time in multiple regions, surpassing D614 when both were present, suggesting G614 S protein virus outcompetes D614 S protein virus. To gain insight into the consequences of the D614G mutation, homology modeling using a multi-template threading mechanism with ab initio structural refinement was performed for a region of the S protein (S591 to N710) spanning the D614G mutation and the S1 furin cleavage site. Molecular models of this region containing D614 or G614 revealed a major difference in secondary structure at the furin domain (RRARS, R682 to S686). The D614 model predicted a random coil structure in the furin domain whereas the G614 model predicted an alpha helix. Critical residues in the cleavage domain of G614 model were found to better align with the PDB structure of a furin inhibitor. Thus, homology modeling studies suggest a potential mechanism whereby the D614G mutation may confer a competitive advantage at the furin binding domain that may contribute to the rise of the D614G SARS-CoV-2 mutant.