A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

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Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

Journal of General Virology ◽

10.1099/jgv.0.001671 ◽

2021 ◽

Vol 102 (11) ◽

Author(s):

Rafaela S. Fontenele ◽

Matias Köhler ◽

Lucas C. Majure ◽

Jesús A. Avalos-Calleros ◽

Gerardo R. Argüello-Astorga ◽

...

Keyword(s):

South America ◽

Rolling Circle Replication ◽

Dna Virus ◽

Single Stranded Dna ◽

Dna Viruses ◽

Circular Dna ◽

Rolling Circle ◽

Rep Protein ◽

Molecular Features ◽

Viral Components

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

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Rolling-Circle Replication of an Animal Circovirus Genome in a Theta-Replicating Bacterial Plasmid in Escherichia coli

Journal of Virology ◽

10.1128/jvi.00655-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8686-8694 ◽

Cited By ~ 39

Author(s):

Andrew K. Cheung

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Mammalian Cells ◽

Unit Length ◽

Release Mechanism ◽

Rolling Circle Replication ◽

Replication Process ◽

Rolling Circle ◽

Rep Protein ◽

Bacterial Plasmid

ABSTRACT A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1Rep/PCV2Cap genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1Cap/PCV2Rep genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE1 Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep′ protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep′ protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

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Structural mechanism of DNA recognition by the p204 HIN domain

Nucleic Acids Research ◽

10.1093/nar/gkab076 ◽

2021 ◽

Vol 49 (5) ◽

pp. 2959-2972

Author(s):

Xiaojiao Fan ◽

Jiansheng Jiang ◽

Dan Zhao ◽

Feng Chen ◽

Huan Ma ◽

...

Keyword(s):

Crystal Structure ◽

Structural Information ◽

Dna Recognition ◽

Binding Mode ◽

Structural Mechanism ◽

Homologous Protein ◽

Dimerization Interface ◽

Inducible Protein ◽

Dsdna Binding ◽

Shed Light

Abstract The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.

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Peptide Aptamers That Bind to a Geminivirus Replication Protein Interfere with Viral Replication in Plant Cells

Journal of Virology ◽

10.1128/jvi.02698-05 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5841-5853 ◽

Cited By ~ 35

Author(s):

Luisa Lopez-Ochoa ◽

Jorge Ramirez-Prado ◽

Linda Hanley-Bowdoin

Keyword(s):

Viral Replication ◽

Dna Cleavage ◽

Hot Spot ◽

Rolling Circle Replication ◽

Viral Dna ◽

Peptide Aptamers ◽

Dna Viruses ◽

Rolling Circle ◽

Random Peptide ◽

N Terminus

ABSTRACT The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.

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Integrating genome sequence and structural data for statistical learning to predict transcription factor binding sites

Nucleic Acids Research ◽

10.1093/nar/gkaa1134 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12604-12617

Author(s):

Pengpeng Long ◽

Lu Zhang ◽

Bin Huang ◽

Quan Chen ◽

Haiyan Liu

Keyword(s):

Genome Sequence ◽

Energy Function ◽

Structural Information ◽

Structural Data ◽

P Values ◽

A Genome ◽

Z Scores ◽

Transcription Regulators ◽

Dna Specificity ◽

Tetracycline Repressor

Abstract We report an approach to predict DNA specificity of the tetracycline repressor (TetR) family transcription regulators (TFRs). First, a genome sequence-based method was streamlined with quantitative P-values defined to filter out reliable predictions. Then, a framework was introduced to incorporate structural data and to train a statistical energy function to score the pairing between TFR and TFR binding site (TFBS) based on sequences. The predictions benchmarked against experiments, TFBSs for 29 out of 30 TFRs were correctly predicted by either the genome sequence-based or the statistical energy-based method. Using P-values or Z-scores as indicators, we estimate that 59.6% of TFRs are covered with relatively reliable predictions by at least one of the two methods, while only 28.7% are covered by the genome sequence-based method alone. Our approach predicts a large number of new TFBs which cannot be correctly retrieved from public databases such as FootprintDB. High-throughput experimental assays suggest that the statistical energy can model the TFBSs of a significant number of TFRs reliably. Thus the energy function may be applied to explore for new TFBSs in respective genomes. It is possible to extend our approach to other transcriptional factor families with sufficient structural information.

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Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

International Journal of Molecular Sciences ◽

10.3390/ijms22147704 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7704

Author(s):

Sayi’Mone Tati ◽

Laleh Alisaraie

Keyword(s):

Binding Affinity ◽

Atp Hydrolysis ◽

Critical Role ◽

Motor Protein ◽

Structural Data ◽

Retrograde Transport ◽

Cytoplasmic Dynein ◽

Motor Domain ◽

Conformational Search ◽

Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

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Targeted Allele Replacement Mutagenesis of Corynebacterium pseudotuberculosis

Applied and Environmental Microbiology ◽

10.1128/aem.01740-10 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3532-3535 ◽

Cited By ~ 2

Author(s):

Caray A. Walker ◽

Willie Donachie ◽

David G. E. Smith ◽

Michael C. Fontaine

Keyword(s):

Corynebacterium Pseudotuberculosis ◽

Sequence Length ◽

Homologous Sequence ◽

Proof Of Concept ◽

Rolling Circle Replication ◽

Content Type ◽

Rolling Circle ◽

Allele Replacement ◽

Marker Free ◽

The Relationship

ABSTRACTA two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations inCorynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free,cp40-deficient mutant ofC. pseudotuberculosiswas constructed.

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Homogeneous Detection of Single Rolling Circle Replication Products

Analytical Chemistry ◽

10.1021/ac034987+ ◽

2004 ◽

Vol 76 (2) ◽

pp. 495-498 ◽

Cited By ~ 48

Author(s):

Gerhard A. Blab ◽

Thomas Schmidt ◽

Mats Nilsson

Keyword(s):

Rolling Circle Replication ◽

Rolling Circle

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Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽

10.1128/mbio.00395-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 14

Author(s):

Matthew J. Belousoff ◽

Zohar Eyal ◽

Mazdak Radjainia ◽

Tofayel Ahmed ◽

Rebecca S. Bamert ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Structural Information ◽

Structural Basis ◽

Common Mechanism ◽

Resistance Mutations ◽

Antimicrobial Drugs ◽

Linezolid Resistance ◽

Drug Modification ◽

Antibiotic Resistance Mutations ◽

Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

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Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases

International Journal of Molecular Sciences ◽

10.3390/ijms22030997 ◽

2021 ◽

Vol 22 (3) ◽

pp. 997

Author(s):

Livija Tušar ◽

Aleksandra Usenik ◽

Boris Turk ◽

Dušan Turk

Keyword(s):

Binding Site ◽

Structural Information ◽

General Rule ◽

Structural Data ◽

Cysteine Proteases ◽

Protein Inhibitors ◽

Active Site Cleft ◽

Living Organisms ◽

And Control ◽

Binding Mechanisms

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

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