Biology subjects

Barford, D.

Publications and source records attributed to Barford, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanism and regulation of Bim1 interactions withthe S. cerevisiae outer kinetochore Ndc80 complex

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an alpha-helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

molecular biology↗

bioRxiv2026

Mechanism and regulation of the Bim1 interaction withthe outer kinetochore Ndc80 complex in S. cerevisiae

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an -helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

cell biology↗

bioRxiv2026

Molecular Mechanism of Condensin II auto-repression by NCAPD3 and activation by M18BP1

Human condensin II is a constitutively nuclear molecular motor that initiates chromosome organization in early mitosis. How condensin II is activated specifically in mitosis remains unknown. Here, we describe the molecular mechanism underlying condensin II auto-repression and activation. By determining multiple structural states of condensin II, we discovered that an autoinhibitory tail within the NCAPD3 subunit (NCAPD3Tail) holds the complex in a conformation that is incompatible with DNA capture. Deletion of NCAPD3Tail spontaneously activates condensin II in cells, illuminating its autoinhibitory role in vivo. We further show this translates to increased loop DNA formation by condensin II in vitro. Direct competition for the NCAPD3Tail binding site on condensin II enables the putative activator protein M18BP1 to liberate a key DNA-binding element in the NCAPH2 N-terminus, enabling DNA capture. Unexpectedly, M18BP1 not only relieves autoinhibition but also directly contributes to DNA organization by forming a positively charged loop that enhances DNA-anchoring by condensin II. Together, these findings reveal a bipartite activation mechanism wherein M18BP1 relieves autorepression, and renders condensin II biochemically competent to form stable DNA loops, ensuring highly stringent regulation of mitotic chromosome formation.

biochemistry↗

bioRxiv2025

Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays

Human kinetochores assemble onto centromeric DNA defined by tandem copies of thousands of the 171 bp -satellite repeat sequence. The centromere-specific CENP-A nucleosome (CENP-ANuc) recruits the inner kinetochore constitutive centromere associated network (CCAN) complex. A previous cryo-EM structure of CCAN bound to a CENP-ANuc reconstituted with a 171 bp -satellite repeat sequence showed how extranucleosomal linker DNA threads through an internal tunnel in the CCAN complex. To understand the higher-order architecture of the inner kinetochore assembled onto -satellite repeat arrays, we have determined cryo-EM structures of CCAN with longer DNA sequences. These include free DNA and single and dimeric -satellite repeats with CENP-A nucleosomes. We show from the structures of CCAN bound to both free DNA and monomeric CENP-ANuc that CCAN engages 65-70 bp of DNA comprising 30-35 bp of an upstream -satellite repeat. This upstream DNA interacts with the histone-fold domain subunits of the CENP-TWSX module in a manner resembling how DNA is wrapped in nucleosomes. A complex of CCAN assembled onto a dimeric -satellite repeat with two CENP-A nucleosomes showed that CCAN can only be accommodated on the linker DNA by unwrapping DNA from the CENP-TWSX module together with 25 bp of DNA from the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of -satellite chromatin containing CENP-ANuc.

molecular biology↗

bioRxiv2025

Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex

During mitosis and meiosis kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation in all eukaryotes, however recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. In this study, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The ten-subunit KMN complex comprises three subcomplexes: Knl1c, Mis12cMtw1c and Ndc80c. We show that -helical motifs in the C-termini of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1 and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two -helices that engage the Mis12cMtw1c head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-CMif2 and CENP-UAme1, reducing their affinity for Mis12cMtw1. Our structure reveals how Aurora BIpl1 phosphorylation of Dsn1AI would release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore. SummaryKinetochore-localised KMN complexes mediate chromosome segregation by attaching chromosomes to spindle microtubules. Cryo-EM, biochemical and genetic experiments reveal conserved underlying mechanisms of complex assembly, auto-inhibition and phospho-regulation that ensure precise control of KMN assembly onto centromeres to enable genome division.

biochemistry↗

bioRxiv2024

Human spermatogenesis leads to a reduced nuclear pore structure and function

Nuclear pore complexes (NPCs) are nuclear gateways which regulate the transit of molecules larger than 40kDa through a Ran-dependent transport1. The somatic human NPC scaffold consists of three stacked rings: the cytoplasmic (CR), the nucleoplasmic (NR), and the inner ring (IR), which define a central channel approximately 55nm wide2. Although many studies have investigated human NPC architecture3, it remains largely unknown how the NPC accommodates the different functions of non-somatic cells. Here, we reveal the in-cell architecture of the human sperm NPC. We show that it exhibits a central channel less than 40nm-wide, outlined exclusively by the IR. This structural alteration is accompanied by a six-fold reduction in nuclear diffusion rate and mis-localization of Ran-dependent transport components. Additionally, we identify a network of septin filaments interconnecting NPCs within the inter-membrane space of the nuclear envelope (NE), suggesting a potential mechanical role in channel constriction. Furthermore, our human tissue imaging data indicate meiosis as a pivotal differentiation stage driving architectural changes to the NPC. Given the critical role of the IR for successful spermatogenesis4,5, our work offers important insight into this fundamental biological process. Our study also demonstrates the power of an integrative approach by combining electron-cryo- tomography, super-resolution-light-microscopy and biochemical analysis, to elucidate macromolecular structure-function relations in human physiological contexts.

cell biology↗

bioRxiv2024

A comparative study of the cryo-EM structures of S. cerevisiae and human anaphase-promoting complex/cyclosome (APC/C)

The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that controls progression through the cell cycle by orchestrating the timely proteolysis of mitotic cyclins and other cell cycle regulatory proteins. Although structures of multiple human APC/C complexes have been extensively studied over the past decade, the S. cerevisiae APC/C has been less extensively investigated. Here, we describe medium resolution structures of three S. cerevisiae APC/C complexes: unphosphorylated apo-APC/C and the ternary APC/CCDH1-substrate complex, and phosphorylated apo-APC/C. Whereas the overall architectures of human and S. cerevisiae APC/C are conserved, as well as the mechanism of CDH1 inhibition by CDK-phosphorylation, specific variations exist, including striking differences in the mechanism of coactivator-mediated stimulation of E2 binding, and the activation of APC/CCDC20 by phosphorylation. In contrast to human APC/C in which coactivator induces a conformational change of the catalytic module APC2:APC11 to allow E2 binding, in S. cerevisiae apo-APC/C the catalytic module is already positioned to bind E2. Furthermore, we find no evidence of a phospho-regulatable auto-inhibitory segment of APC1, that in the unphosphorylated human APC/C, sterically blocks the CDC20C-box binding site of APC8. Thus, although the functions of APC/C are conserved from S. cerevisiae to humans, molecular details relating to their regulatory mechanisms differ.

molecular biology↗

bioRxiv2024

Development of D-box peptides to inhibit the Anaphase Promoting Complex/Cyclosome

E3 ubiquitin ligases engage their substrates via degrons - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are hard to drug via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the Destruction box (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20- peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/CCdc20 and are more potent than the small molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially also for harnessing APC/C for targeted protein degradation.

biochemistry↗

bioRxiv2024

Molecular mechanism of Mad2 conformational conversion promoted by the Mad2-interaction motif of Cdc20

During mitosis, unattached kinetochores trigger the spindle assembly checkpoint by promoting assembly of the mitotic checkpoint complex, a heterotetramer comprising Mad2, Cdc20, BubR1 and Bub3. Critical to this process is the kinetochore-mediated catalysis of an intrinsically slow conformational conversion of Mad2 from an open (O-Mad2) inactive state to a closed (C-Mad2) active state bound to Cdc20. These Mad2 conformational changes involve substantial remodelling of the N-terminal {beta}1 strand and C-terminal {beta}7/{beta}8 hairpin. In vitro, the Mad2- interaction motif (MIM) of Cdc20 (Cdc20MIM) triggers rapid conversion of O- to C-Mad2, effectively removing the kinetic barrier for MCC assembly. How Cdc20MIM directly induces Mad2 conversion remains unclear. In this study we demonstrate that the Cdc20MIM-binding site is inaccessible in O-Mad2. Time-resolved NMR and molecular dynamics simulations show how Mad2 conversion involves sequential conformational changes of flexible structural elements in O-Mad2, orchestrated by Cdc20MIM. Conversion is initiated by the {beta}7/{beta}8 hairpin of O-Mad2 transiently unfolding to expose a nascent Cdc20MIM-binding site. Engagement of Cdc20MIM to this site promotes release of the {beta}1 strand. We propose that initial conformational changes of the {beta}7/{beta}8 hairpin allows binding of Cdc20MIM to a transient intermediate state of Mad2, thereby lowering the kinetic barrier to Mad2 conversion.

biochemistry↗

bioRxiv2023

New structural features of the APC/C revealed by high resolution cryo-EM structures of apo-APC/C and the APC/C CDH1:EMI1 complex

The multi-subunit anaphase-promoting complex/cyclosome (APC/C) is a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins and other cell cycle regulatory proteins for degradation. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications, and its inhibitory binding partners EMI1 and the mitotic checkpoint complex (MCC). In this study, we took advantage of the latest developments in cryo-electron microscopy (cryo-EM) to determine the structures of human APC/CCDH1:EMI1 and apo-APC/C at 2.9 [A] and 3.2 [A] resolution, respectively, providing novel insights into the regulation of APC/C activity. The high-resolution maps allowed the unambiguous assignment of a previously unassigned -helix to the N-terminus of CDH1 (CDH11) in the APC/CCDH1:EMI1 ternary complex. We also identified a novel zinc-binding module in APC2 that confers structural stability to APC2, and we confirmed the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps, we were able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that likely play a role in proper APC/C assembly and regulation of its activity.

cell biology↗

bioRxiv2023

Structure of the human outer kinetochore KMN network complex

Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network, which comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C. The redundancy and strength of inter-subunit connections explains how KMN withstands strong forces applied during chromosome segregation. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our work provides a molecular mechanism for how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation would restrict outer kinetochore assembly to functional centromeres during cell division.

cell biology↗

bioRxiv2023

Mechanism of outer kinetochore assembly on microtubules and its regulation by mitotic error correction

Kinetochores couple chromosomes to the mitotic spindle and transduce the energy of microtubule depolymerization to segregate the genome during cell division. Kinetochore - microtubule attachments are often initially erroneous and subject to a mitotic error correction (EC) mechanism that drives their turnover until biorientation is achieved. How this is accomplished and regulated, and how kinetochore-mediated chromosome segregation occurs at a molecular level remain major outstanding questions. Here we describe the cryo-electron microscopy (cryo-EM) structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. We observe coordinated interactions of the outer kinetochore complexes through multiple interfaces, in addition to a short staple within the Dam1 subunit that facilitates Dam1c ring assembly. Perturbation of these interfaces results in loss of yeast viability. Force-rupture assays indicated this is a consequence of substantial reductions in kinetochore - microtubule binding strength. EC-mediated phosphorylation of Ndc80c-Dam1c interfaces would drive complex disassembly, whereas Dam1 staple phosphorylation would promote Dam1c ring disassembly, explaining how kinetochore - microtubule attachments are destabilized and reset by the EC mechanism. One-Sentence SummaryPhosphorylation disrupts the outer kinetochore to regulate kinetochore-microtubule attachments in mitotic error correction.

molecular biology↗

bioRxiv2022

Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere

SummaryThe point centromere of budding yeast specifies assembly of the large multi-subunit kinetochore complex. By direct attachment to the mitotic spindle, kinetochores couple the forces of microtubule dynamics to power chromatid segregation at mitosis. Kinetochores share a conserved architecture comprising the centromere-associated inner kinetochore CCAN (constitutive centromere-associated network) complex and the microtubule-binding outer kinetochore KMN network. The budding yeast inner kinetochore additionally includes the centromere-binding CBF1 and CBF3 complexes. Here, we reconstituted the complete yeast inner kinetochore complex assembled onto the centromere-specific CENP-A nucleosome (CENP-ANuc) and determined its structure using cryo-EM. This revealed a central CENP-ANuc, wrapped by only one turn of DNA, and harboring extensively unwrapped DNA ends. These free DNA duplexes function as binding sites for two CCAN protomers, one of which entraps DNA topologically and is positioned precisely on the centromere by the sequence-specific DNA-binding complex CBF1. The CCAN protomers are connected through CBF3 to form an arch-like configuration, binding 150 bp of DNA. We also define a structural model for a CENP-ANuc-pathway to the outer kinetochore involving only CENP-QU. This study presents a framework for understanding the basis of complete inner kinetochore assembly onto a point centromere, and how it organizes the outer kinetochore for robust chromosome attachment to the mitotic spindle.

biochemistry↗

bioRxiv2022

Structural basis for CDC42 and RAC activation by the dual specificity GEF DOCK10

Dedicator of cytokinesis 10 (DOCK10) is a guanine nucleotide exchange factor (GEF) belonging to the DOCK family that activate RHO GTPases. DOCK10 controls amoeboid migration and IL-4 induced B cell activation. Recent structural studies of the catalytic DHR2 domains of DOCK-family GEFs DOCK2 and DOCK9 in the presence of nucleotide-free RAC and CDC42 respectively, have revealed a conserved mechanism of nucleotide exchange distinct from the large family of Dbl homology (DH) GEFs. Less is known from a biochemical and structural perspective, about DOCK10. We have determined the crystal structure of the apo-state of the DHR2 domain of DOCK10 (DOCK10DHR2), which provides the first structural model for the apo-state of a DHR2 domain for the entire DOCK family. It demonstrates the conformational changes within DOCK10DHR2 on engaging the GTPase, and associated induced conformational changes of the GTPase required to stimulate nucleotide release. We also report on the unexpected dual specificity of DOCK10DHR2 which directly interacts with RAC and CDC42 and induces the nucleotide exchange of both in vitro. This dual specificity is unique to DOCK10 within its evolutionarily related D subfamily. Structural studies of DOCK10DHR2 in complex with RAC revealed an intriguing 2:1 stoichiometry between DOCK10DHR2 and the GTPase, that differs from the canonical 1:1 stoichiometry for all other known DOCK-GTPase complexes. This was confirmed in solution using a variety of biophysical techniques. The identification of a novel mode of interaction between DOCK10DHR2 and the RAC GTPase provides new insights into how DOCK family GEFs discriminate between CDC42, RAC and RHOA.

biochemistry↗

bioRxiv2022

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase- promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC is a complex of Mad2, Cdc20 and BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a catalytic Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O- Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires conversion of O-Mad2 to C-Mad2 and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. In this study we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C- terminal domain of Mad1 (Mad1CTD). We additionally identified a folded state of the Mad1:Mad2 complex that suggests how the Cdc20:Mad1CTD interaction brings the Mad2- interacting motif (MIM) of Cdc20 (Cdc20MIM) into close proximity with O-Mad2. We also show that Cdc20MIM is sufficient to catalyse Mad2 conversion and its entrapment by the Mad2 safety- belt. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

biochemistry↗

bioRxiv2022

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an [~]171 bp -satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto -satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the -satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the -satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces. One-Sentence SummaryThe human inner kinetochore CCAN complex tightly grips the linker DNA of the -satellite CENP-A nucleosome.

biochemistry↗

bioRxiv2021

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

CDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.

cancer biology↗

bioRxiv2020

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

molecular biology↗