Thumb Domain Research Articles

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

Molecular effects of site-specific phosphate-methylated primer on the structure and motions of Taq DNA polymerase

Polymerase chain reaction (PCR) is a powerful molecular biology assay for gene detection and quantification. Conventional DNA primers for PCR often suffer from poor sensitivity in specific gene detection. Recently, oligonucleotides containing methyl phosphotriester (MPTE-DNA) have been developed with enhanced DNA hybridization and improved gene detection sensitivity. Yet, site-specific MPTE-modifications on DNA primers have been reported to affect PCR amplification efficiencies while the detailed mechanism remains elusive. Here, we utilized molecular dynamics (MD) simulation to examine the effects of site-specific MPTE-modified primers on the structure and motions of DNA/Taq polymerase complexes. All tested MPTE-DNA/Taq complexes exhibited RMSD values below 2 Å, indicating insignificant effects of all methylation sites on the complex stability. The energetic and hydrogen-bonding analyses suggest minor effects of methylation at t-3, t-4, t-6, and t-7 positions on the DNA−Taq interaction. Principal component analyses further reveal that only t-3, and t-7 methylations preserve the motions of the Taq thumb domain. The site-specific methylation affects the interaction between DNA and the surrounding protein residues, resulting in allosteric-like effects on the DNA/Taq complex. The MD data complement the best experimentally observed PCR efficacies for the t-3 and t-7 positions among all tested MPTE-primers. The unveiled molecular insights can benefit the design of novel PCR primers for improving genetic testing platforms.

Investigation of Antiviral Potential of Food Carotenoids and Apocarotenoids against RNA-dependent RNA Polymerase of Hepatitis C Virus

Hepatitis C disease have been a global health threat and affects a significant portion of world population. Hepatitis C have also been a silent health threat for Turkiye, where there are around half million people infected with Hepatitis C Virus (HCV). Disease burden and mortality are expected to increase gradually in the next 20 years in Turkiye. Unavailability of enough data on the currently-available drugs in routine clinical practice, their side effects and interactions with other drugs, and their efficacies on the less common genotypes indicates the necessity of alternative treatment options. Natural products from herbal and medicinal plants can indeed provide an alternative as being drug-like dietary supplements. In particular, the carotenoids and apocarotenoids are underexplored in their antiviral potential, including anti-HCV activities. Therefore, we focused on the virtual screening of various carotenoids and apocarotenoids against the RNA-dependent RNA polymerase (RdRp) of HCV. Molecular docking experiments showed strong binding affinities of the ligands to both palm and thumb domains of RdRp of HCV. In fact, some of them such as neoxanthin, crocin, canthaxanthin and cryptoflavin bound quite strongly to both domains compared to native ligands and current antiviral drugs. MD simulation for neoxanthin-RdRp complex confirmed the stability of the ligand within the binding cavity of RdRp throughout 100 ns simulation. This clearly indicated the potential of carotenoids, specifically neoxanthin, as RdRp inhibitor in treating HCV. Thus, this study not only discovered anti-HCV drug candidates with the properties of easy-to-access and low cost, but also paved the way for the development of carotenoid or apocarotenoid based dietary supplement candidates for the prevention and treatment of HCV.

Targeting Emerging RNA Viruses by Engineered Human Superantibody to Hepatitis C Virus RNA-Dependent RNA Polymerase.

RNA-dependent RNA polymerase (RdRp) is a unique and highly conserved enzyme across all members of the RNA virus superfamilies. Besides, humans do not have a homolog of this protein. Therefore, the RdRp is an attractive target for a broadly effective therapeutic agent against RNA viruses. In this study, a formerly generated cell-penetrating human single-chain antibody variable fragment (superantibody) to a conformational epitope of hepatitis C virus (HCV) RdRp, which inhibited the polymerase activity leading to the HCV replication inhibition and the host innate immunity restoration, was tested against emerging/reemerging RNA viruses. The superantibody could inhibit the replication of the other members of the Flaviviridae (DENV serotypes 1−4, ZIKV, and JEV), Picornaviridae (genus Enterovirus: EV71, CVA16), and Coronaviridae (genus Alphacoronavirus: PEDV, and genus Betacoronavirus: SARS-CoV-2 (Wuhan wild-type and the variants of concern), in a dose-dependent manner, as demonstrated by the reduction of intracellular viral RNAs and numbers of the released infectious particles. Computerized simulation indicated that the superantibody formed contact interfaces with many residues at the back of the thumb domain (thumb II site, T2) of DENV, ZIKV, JEV, EV71, and CVA16 and fingers and thumb domains of the HCV and coronaviruses (PEDV and SARS-CoV-2). The superantibody binding may cause allosteric change in the spatial conformation of the enzyme and disrupt the catalytic activity, leading to replication inhibition. Although the speculated molecular mechanism of the superantibody needs experimental support, existing data indicate that the superantibody has high potential as a non-chemical broadly effective anti-positive sense-RNA virus agent.

Structural Analysis of Monomeric RNA-Dependent Polymerases Revisited.

In the past few years, our understanding of the RNA virosphere has changed dramatically due to the growth and spurt of metagenomics, exponentially increasing the number of RNA viral sequences, and providing a better understanding of their range of potential hosts. As of today, the only conserved protein among RNA viruses appears to be the monomeric RNA-dependent RNA polymerase. This enzyme belongs to the right-hand DNA-and RNA polymerases, which also includes reverse transcriptases and eukaryotic replicative DNA polymerases. The ubiquity of this protein in RNA viruses makes it a unique evolutionary marker and an appealing broad-spectrum antiviral target. In this work pairwise structural comparisons of viral RdRps and RTs were performed, including tertiary structures that have been obtained in the last few years. The resulting phylogenetic tree shows that the RdRps from (+)ss- and dsRNA viruses might have been recruited several times throughout the evolution of mobile genetic elements. RTs also display multiple evolutionary routes. We have identified a structural core comprising the entire palm, a large moiety of the fingers and the N-terminal helices of the thumb domain, comprising over 300 conserved residues, including two regions that we have named the “knuckles” and the “hypothenar eminence”. The conservation of an helix bundle in the region preceding the polymerase domain confirms that (−)ss and dsRNA Reoviruses’ polymerases share a recent ancestor. Finally, the inclusion of DNA polymerases into our structural analyses suggests that monomeric RNA-dependent polymerases might have diverged from B-family polymerases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00239-022-10059-z.

Structure of active human telomerase with telomere shelterin protein TPP1.

Human telomerase is a RNA-protein complex that extends the 3' end of linear chromosomes by synthesizing multiple copies of the telomeric repeat TTAGGG1. Its activity is a determinant of cancer progression, stem cell renewal and cellular aging2-5. Telomerase is recruited to telomeres and activated for telomere repeat synthesis by the telomere shelterin protein TPP16,7. Human telomerase has a bilobal structure with a catalytic core ribonuclear protein and a H and ACA box ribonuclear protein8,9. Here we report cryo-electron microscopy structures of human telomerase catalytic core of telomerase reverse transcriptase (TERT) andtelomerase RNA (TER (also known as hTR)), andof telomerase with the shelterin protein TPP1. TPP1 forms a structured interface with the TERT-unique telomerase essential N-terminal domain (TEN) and the telomerase RAP motif (TRAP) that are unique to TERT, and conformational dynamics of TEN-TRAP are damped upon TPP1 binding, defining the requirements for recruitment and activation. The structures further reveal that the elements of TERT and TER that are involved in template and telomeric DNA handling-including the TEN domain and the TRAP-thumb helix channel-are largely structurally homologous to those in Tetrahymena telomerase10, and provide unique insights into the mechanism of telomerase activity. The binding site of the telomerase inhibitor BIBR153211,12 overlaps a critical interaction between the TER pseudoknot and the TERT thumb domain. Numerous mutations leading to telomeropathies13,14 are located at the TERT-TER and TEN-TRAP-TPP1 interfaces, highlighting the importance of TER-TERT and TPP1 interactions for telomerase activity, recruitment and as drug targets.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2.

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

An induced-fit de novo initiation mechanism suggested by a pestivirus RNA-dependent RNA polymerase.

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a ‘priming element’ (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665–680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar ‘induced-fit’ mechanisms may be commonly taken by PE-containing de novo viral RdRPs.

Intersubunit polar interactions within extracellular domains modulate ENaC gating

Epithelial Na+ channel (ENaC) gating is modulated by a variety of extracellular factors that induce conformational changes within the channel. Previous studies of ENaCs and homologous acid sensing ion channels (ASICs) have implicated the palm, thumb and other extracellular domains in regulating ENaC gating. However, structural transitions associated with changes in channel gating are not well understood. Based on our previous studies and the structures of ENaC and ASIC1, we hypothesized that specific inter-domain interactions at subunit interfaces have important roles in modulating ENaC gating. In this study, we used cross-linking to identify intersubunit interactions involving the palm domains. Selected residues of palm domain b10 strand, b11-b12 linker, and thumb domain a4 helix were individually mutated to Cys. ENaC subunits (a, b, and g) with either zero, one or two introduced Cys residues were co-expressed in Xenopus oocytes. The reducing agent DTT was externally applied to assess for the spontaneous formation of a disulfide bridge between two introduced Cys residues. The oxidizing reagent H2O2 was used to induce a disulfide bridge between two Cys residues. We found that bE499C within b11-b12 linker and aN510C within b10 strand form both spontaneous and induced disulfide bond, based on the inhibitory effect of DTT and the stimulatory effect of H2O2 on the double mutant (aN510CbE499Cg). The DTT effect was smaller in magnitude than the H2O2 effect. Both DTT and H2O2 did not dramatically alter the currents of WT, aN510Cbg or abE499Cg channels. Interestingly, bE499C formed a cross-link with aQ441C within the a4 helix of thumb domain in the absence of an oxidizing reagent. Oocytes expressing aQ441CbE499Cg showed significantly smaller currents than WT and the single mutants. DTT dramatically increased its currents. Although H2O2 showed no significant effect on the double mutant, it reversed the strong activation of DTT. Our results suggest that crosslinking αN510C in b10 strand and bE499C in b11-b12 linker activates ENaC by stabilizing open state while crosslinking αQ441C in thumb domain with bE499 locks channel in a low activity state. Our results suggest that bE499 interacts with either aN510 in the palm domain or aQ441 in the thumb domain through hydrogen bonds. These interactions serve as a switch, tuning ENaC open probability. We conclude that intersubunit polar interactions between two adjacent palm domains and between thumb and palm domains modulate ENaC gating.

Prophylactic and therapeutic roles of Glycyrrhiza glabra in the prevention and treatment of COVID19

Introduction ‐COVID19 has become one of the world's leading problems. The severe acute respiratory syndrome coronavirus (SARS‐CoV)‐2 enters the human body through droplet transmission and oroanal route. The pathological manifestations of SARS‐CoV‐2 is mediated through key proteins involved in viral entry into the host and replication of the virus within the host. Specifically, the RNA Dependent RNA Polymerase (RdRp) employs a replicative function in multiplying and transmitting the virus, aided by other nsps, nsp7 and 8 forming functional complex; similarly, the spike protein is necessary for the virus to enter the host by binding to ACE receptors. Given the limited available treatments and urgency for the development of prophylactic agents and therapeutic drugs for the control of SARS‐CoV‐2 and treatment of COVID19, traditional treatment methods using natural resources are decisive. To address the pandemic, a drug targeting the function of these key proteins and protein complexes will be potentially more successful and effective. Use of medically important plants with antiviral activity including, Glycyrrhiza glabra (G glabra) and isolation of potential natural active ingredients is more beneficial and effective. Here we elucidated the potential interactions of known phytochemicals from G glabra and key SARS‐CoV‐2 proteins.Methods ‐A molecular docking algorithm, PatchDock, was used to study the interactions between viral target proteins and phytochemicals of G. glabra. The molecular display program, Chimera was used to display the interactions between the phytochemicals of G. glabra and viral proteins.Results ‐Of the 20 reported compounds (GG1 ‐ GG20) that we used for our studies, 13 of the compounds have effectively bound to inhibit the SARS‐CoV‐2 proteins, nsp7/8/12 complex including RdRp and/or spike protein. Specifically, the compounds GG3 and GG4 achieved lower ACE scores than ‐100 binding to all three key proteins of the SARS‐COV‐2. Compound GG4 binds to the Spike Protein that mediate binding of SARS‐COV‐2 to the ACE Receptor of host cell, thereby inhibiting the entry of virus. GG4 also binds to close to the active site of nsp‐complex. However, GG3 binds close to the active site of the RdRp where the thumb, finger, and palm domains meet. GG3 also binds close to the nsp7/8/12 complex in which chains 7 and 12 join together, forming a more effective complex to inhibit the viral replication. Our additional docking studies of phytochemicals of G. glabra and viral proteins will show effective binding of phytochemicals with viral proteins that inhibit either the entry and/or replication and transmission of the SARS‐CoV‐2.Conclusion –In conclusion, GG3 and GG4 from Glycyrrhiza glabra have demonstrated the ability to prevent the entry and replication of SARS‐COV‐2 proteins by inhibiting the functions of Spike and RdRp proteins in silico.

Efficacy of Tinospora cordifolia in treating SARS‐CoV‐2: in silico studies

IntroductionSARS‐CoV‐2 has struck as a major global pandemic causing COVID19 with acute respiratory distress syndrome (ARDS) and other respiratory diseases gradually affecting multiple organs. Currently, SARS‐CoV‐2 is affecting over 85.1 million global cases of SARS‐CoV‐2 (20.7 millions in US) infections with 1.84 million (0.352 million in US) deaths globally. SARS‐CoV‐2 genome is 30kb in size and makes 29 proteins including key Spike protein (SP) and RNA dependent RNA polymerase (RdRP). The SP facilitates the entry of the virus into human host cells. Interactions of potential drugs with the SP can prevent or inhibit the entry of the virus into the host cells by blocking the interaction. Similarly, the non‐structural proteins (nsp) 7 and 8 facilitate the function of the RdRp/nsp12 that is essential for the replication of viral genome leading to multiply and spread of the virus within and from the host. In this study, we have selected 26 known phytochemicals (TC1 ‐ TC26) from medicinally important Ayurvedic plant, Tinospora cordifolia (TC), to study the interactions of these phytochemicals with key SARS‐CoV‐2 proteins using in silico methods. We have identified promising interactions between TC phytochemicals, and the key SARS‐CoV2 proteins that can potentially block the viral entry and/or replication of SARS‐CoV‐2 thereby preventing and treating COVID19.MethodsA molecular docking algorithm, PatchDock, was used to study the interactions between target proteins and phytochemicals of TC. To filter out the redundant docking predictions resulting from the patch matching system, a RMSD clustering calculation is used. The molecular display program, Chimera, was used to display the interactions between the phytochemicals and SARS‐CoV‐2 viral proteins.ResultsOf the 26 compounds tested (TC1 ‐ TC26), 11 showed effective binding to inhibit the SARS‐CoV‐2 proteins, SP or nsp7/8/12 complex including RdRp. The effectiveness of the docking compounds was measured in terms of atomic contact energy (ACE) Scores and rankings based on clusters. The compounds TC1, TC4, TC10 and TC14 achieved lower scores (of binding) less than ‐100 showing potential binding to all three viral proteins/complexes, SP, RdRp and nsp7/8/12. Further, analysis indicates that TC4 and TC10 are binding remarkably close to the active site of the SP that binds to the ACE2 receptor of host cells, providing evidence for effective inhibition of the SP. TC1 and TC14 binds between the N‐terminal, palm domain, and thumb domain of the RdRp, creating an interaction inside the active site, substantially inhibiting its critical replication function.ConclusionFor the first time, we present in silico evidence that TC4 and TC10, and TC1 and TC14 potentially impair the entry and replication of SARS‐COV‐2 proteins respectively by inhibiting the functions of Spike and RdRp/nsp7‐8/12 proteins respectively.

Mambalgin-1 pain-relieving peptide locks the hinge between α4 and α5 helices to inhibit rat acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are proton-gated cationic channels involved in pain and other processes, underscoring the potential therapeutic value of specific inhibitors such as the three-finger toxin mambalgin-1 (Mamb-1) from snake venom. A low-resolution structure of the human-ASIC1a/Mamb-1 complex obtained by cryo-electron microscopy has been recently reported, implementing the structure of the chicken-ASIC1/Mamb-1 complex previously published. Here we combine structure-activity relationship of both the rat ASIC1a channel and the Mamb-1 toxin with a molecular dynamics simulation to obtain a detailed picture at the level of side-chain interactions of the binding of Mamb-1 on rat ASIC1a channels and of its inhibition mechanism. Fingers I and II of Mamb-1 but not the core of the toxin are required for interaction with the thumb domain of ASIC1a, and Lys-8 of finger I potentially interacts with Tyr-358 in the thumb domain. Mamb-1 does not interfere directly with the pH sensor as previously suggested, but locks by several contacts a key hinge between α4 and α5 helices in the thumb domain of ASIC1a to prevent channel opening. Our results provide an improved model of inhibition of mammalian ASIC1a channels by Mamb-1 and clues for further development of optimized ASIC blockers.

Structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7and AcrIF14.

CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC50) = 9.7 ± 0.5 μM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC50 = 1.7 ± 0.4 μM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

Vulnerable targets in HIV-1 Pol for attenuation-based vaccine design

Identification of viral immune escape mutations that compromise HIV's ability to replicate may aid rational attenuation-based vaccine design. Previously we reported amino acids associated with altered viral replication capacity (RC) from a sequence-function analysis of 487 patient-derived RT-integrase sequences. In this study, site-directed mutagenesis experiments were performed to validate the effect of these mutations on RC. Viral reverse transcripts were measured by quantitative PCR and structural modelling was performed to gain further insight into the effect of reverse transcriptase (RT) mutations on reverse transcription. RT-integrase variants in or flanking cytotoxic T cell epitopes in the RT palm (158S), RT thumb (241I and 257V) and integrase catalytic core domain (124N) were confirmed to significantly reduce RC. RT mutants showed a delayed initiation of viral DNA synthesis. Structural models provide insight into how these attenuating RT mutations may affect amino acid interactions in the helix clamp, primer grip and catalytic site regions.

ASFV DNA polymerase extends recessed DNAs with catalytic efficiencies outperforming those exerted on gapped DNA substrates

The DNA polymerase from african swine fever virus (ASFV Pol X), lacking both 8 kDa and thumb domains, is the smallest enzyme featuring competence in DNA extension. Here we show that ASFV Pol X features poor filling activity of DNA gaps consisting of 15 bases, and exerts a more efficient action at the expense of DNA substrates containing a recessed end of equal length. We also show that shortening the recessed end of DNA substrates decreases the rate of DNA elongation catalysed by ASFV Pol X. Finally, by means of stopped-flow experiments we were able to determine that DNA binding is a step responsible for restraining the efficiency of ASFV Pol X catalytic action.

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3′-5′ exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

How a B family DNA polymerase has been evolved to copy RNA

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.

Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication.