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1. The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid

   https://doi.org/10.1128/JVI.00883-21  

   Ogunbunmi, Elizabeth T.; Roznowski, Aaron P.; Fane, Bentley A. (August 2021, Journal of Virology)

   Sandri-Goldin, Rozanne M. (Ed.)

   ABSTRACT Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface. 

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2. Structural predictions of protein–DNA binding: MELD-DNA

   https://doi.org/10.1093/nar/gkad013  

   Esmaeeli, Reza; Bauzá, Antonio; Perez, Alberto (February 2023, Nucleic Acids Research)

   Abstract Structural, regulatory and enzymatic proteins interact with DNA to maintain a healthy and functional genome. Yet, our structural understanding of how proteins interact with DNA is limited. We present MELD-DNA, a novel computational approach to predict the structures of protein–DNA complexes. The method combines molecular dynamics simulations with general knowledge or experimental information through Bayesian inference. The physical model is sensitive to sequence-dependent properties and conformational changes required for binding, while information accelerates sampling of bound conformations. MELD-DNA can: (i) sample multiple binding modes; (ii) identify the preferred binding mode from the ensembles; and (iii) provide qualitative binding preferences between DNA sequences. We first assess performance on a dataset of 15 protein–DNA complexes and compare it with state-of-the-art methodologies. Furthermore, for three selected complexes, we show sequence dependence effects of binding in MELD predictions. We expect that the results presented herein, together with the freely available software, will impact structural biology (by complementing DNA structural databases) and molecular recognition (by bringing new insights into aspects governing protein–DNA interactions). 

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3. Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System

   https://doi.org/10.1128/jvi.00990-22  

   Lee, Hyunwook; Baxter, Alexis J.; Bator, Carol M.; Fane, Bentley A.; Hafenstein, Susan L. (November 2022, Journal of Virology)

   Frappier, Lori (Ed.)

   ABSTRACT Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo . Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8’s C-terminus and a portion of VP1’s N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae . The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa. 

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4. HybridDBRpred: improved sequence-based prediction of DNA-binding amino acids using annotations from structured complexes and disordered proteins

   https://doi.org/10.1093/nar/gkad1131  

   Zhang, Jian; Basu, Sushmita; Kurgan, Lukasz (December 2023, Nucleic Acids Research)

   Abstract Current predictors of DNA-binding residues (DBRs) from protein sequences belong to two distinct groups, those trained on binding annotations extracted from structured protein-DNA complexes (structure-trained) vs. intrinsically disordered proteins (disorder-trained). We complete the first empirical analysis of predictive performance across the structure- and disorder-annotated proteins for a representative collection of ten predictors. Majority of the structure-trained tools perform well on the structure-annotated proteins while doing relatively poorly on the disorder-annotated proteins, and vice versa. Several methods make accurate predictions for the structure-annotated proteins or the disorder-annotated proteins, but none performs highly accurately for both annotation types. Moreover, most predictors make excessive cross-predictions for the disorder-annotated proteins, where residues that interact with non-DNA ligand types are predicted as DBRs. Motivated by these results, we design, validate and deploy an innovative meta-model, hybridDBRpred, that uses deep transformer network to combine predictions generated by three best current predictors. HybridDBRpred provides accurate predictions and low levels of cross-predictions across the two annotation types, and is statistically more accurate than each of the ten tools and baseline meta-predictors that rely on averaging and logistic regression. We deploy hybridDBRpred as a convenient web server at http://biomine.cs.vcu.edu/servers/hybridDBRpred/ and provide the corresponding source code at https://github.com/jianzhang-xynu/hybridDBRpred. 

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5. Salt bridge dynamics in protein/DNA recognition: a comparative analysis of Elk1 and ETV6

   https://doi.org/10.1039/D1CP01568K  

   Vo, Tam D.; Schneider, Amelia L.; Wilson, W. David; Poon, Gregory M. (January 2021, Physical Chemistry Chemical Physics)

   null (Ed.)

   Electrostatic protein/DNA interactions arise from the neutralization of the DNA phosphodiester backbone as well as coupled exchanges by charged protein residues as salt bridges or with mobile ions. Much focus has been and continues to be paid to interfacial ion pairs with DNA. The role of extra-interfacial ionic interactions, particularly as dynamic drivers of DNA sequence selectivity, remain poorly known. The ETS family of transcription factors represents an attractive model for addressing this knowledge gap given their diverse ionic composition in primary structures that fold to a tightly conserved DNA-binding motif. To probe the importance of extra-interfacial salt bridges in DNA recognition, we compared the salt-dependent binding by Elk1 with ETV6, two ETS homologs differing markedly in ionic composition. While both proteins exhibit salt-dependent binding with cognate DNA that corresponds to interfacial phosphate contacts, their nonspecific binding diverges from cognate binding as well as each other. Molecular dynamics simulations in explicit solvent, which generated ionic interactions in agreement with the experimental binding data, revealed distinct salt-bridge dynamics in the nonspecific complexes formed by the two proteins. Impaired DNA contact by ETV6 resulted in fewer backbone contacts in the nonspecific complex, while Elk1 exhibited a redistribution of extra-interfacial salt bridges via residues that are non-conserved between the two ETS relatives. Thus, primary structure variation in ionic residues can encode highly differentiated specificity mechanisms in a highly conserved DNA-binding motif. 

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