Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.

Update of bioRxiv. 2022 May 12;:

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

Many single-stranded, positive-sense RNA viruses regulate assembly of their infectious virions by forming multiple, cognate coat protein (CP)-genome contacts at sites termed Packaging Signals (PSs). We have determined the secondary structures of the bacteriophage MS2 ssRNA genome (gRNA) frozen in defined states using constraints from X-ray synchrotron footprinting (XRF). Comparison of the footprints from phage and transcript confirms the presence of multiple PSs in contact with CP dimers in the former. This is also true for a virus-like particle (VLP) assembled around the gRNA in vitro in the absence of the single-copy Maturation Protein (MP) found in phage. Since PS folds are present at many sites across gRNA transcripts, it appears that this genome has evolved to facilitate this mechanism of assembly regulation. There are striking differences between the gRNA-CP contacts seen in phage and the VLP, suggesting that the latter are inappropriate surrogates for aspects of phage structure/function. Roughly 50% of potential PS sites in the gRNA are not in contact with the protein shell of phage. However, many of these sit adjacent to, albeit not in contact with, PS-binding sites on CP dimers. We hypothesize that these act as PSs transiently during assembly but subsequently dissociate. Combining the XRF data with PS locations from an asymmetric cryo-EM reconstruction suggests that the genome positions of such dissociations are non-random and may facilitate infection. The loss of many PS-CP interactions towards the 3' end of the gRNA would allow this part of the genome to transit more easily through the narrow basal body of the pilus extruding machinery. This is the known first step in phage infection. In addition, each PS-CP dissociation event leaves the protein partner trapped in a non-lowest free-energy conformation. This destabilizes the protein shell which must disassemble during infection, further facilitating this stage of the life-cycle.

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA's are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR.

Membrane protein complexes are crucial for a large variety of biological functions which are mainly dictated by their binding affinity. Due to the intricate nature of their structure, however, the binding affinity of membrane proteins is less explored compared to globular proteins. Mutations in these complexes affect their binding affinity, as well as impair critical functions, and may lead to diseases. Although experimental binding affinity data have expanded in the literature, they are dispersed and it is necessary to compile them into a reliable and comprehensive database. Hence, we developed MPAD (Membrane Protein complex binding Affinity Database), which contains experimental binding affinities of membrane protein-protein complexes and their mutants along with sequence, structure, and functional information, membrane-specific features, experimental conditions, as well as literature information. MPAD has an easy-to-use interface and options to build search queries, display, sort, download, and upload the data are among the other features available to users. This database can be used to understand the factors influencing the binding affinity in membrane proteins when compared to globular proteins as well as the impact of mutations on binding affinity, which may have potential applications to structure-based drug design. MPAD can be freely accessed at https://web.iitm.ac.in/bioinfo2/mpad.

Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, binding receptors and triggering secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide's hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism the signal peptide delays the formation of early initial foldons thus altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein's native core exploit their structural dynamics to influence the folding landscape.

Found in all domains of life, transporters belonging to the LeuT-fold class mediate the import and exchange of hydrophilic and charged compounds such as amino acids, metals, and sugar molecules. Nearly two decades of investigations on the eponymous bacterial transporter LeuT have yielded a library of high-resolution snapshots of its conformational cycle linked by solution-state experimental data obtained from multiple techniques. In parallel, its topology has been observed in symporters and antiporters characterized by a spectrum of substrate specificities and coupled to gradients of distinct ions. Here we review and compare mechanistic models of transport for LeuT, its well-studied homologs, as well as functionally distant members of the fold, emphasizing the commonalities and divergences in alternating access and the corresponding energy landscapes. Our integrated summary illustrates how fold conservation, a hallmark of the LeuT fold, coincides with divergent choreographies of alternating access that nevertheless capitalize on recurrent structural motifs. In addition, it highlights the knowledge gap that hinders the leveraging of the current body of research into detailed mechanisms of transport for this important class of membrane proteins.

Histone chaperones, which constitute an interaction and functional network involved in all aspects of histone metabolism, have to date been identified only in eukaryotes. The Epstein-Barr virus tegument protein BKRF4 is a histone-binding protein that engages histones H2A-H2B and H3-H4, and cellular chromatin, inhibiting the host DNA damage response. Here, we identified BKRF4 as a bona fide viral histone chaperone whose histone-binding domain (HBD) forms a co-chaperone complex with the human histone chaperone ASF1 in vitro. We determined the crystal structures of the quaternary complex of the BKRF4 HBD with human H3-H4 dimer and the histone chaperone ASF1b and the ternary complex of the BKRF4 HBD with human H2A-H2B dimer. Through structural and biochemical studies, we elucidated the molecular basis for H3-H4 and H2A-H2B recognition by BKRF4. We also revealed two conserved motifs, D/EL and DEF/Y/W, within the BKRF4 HBD, which may represent common motifs through which histone chaperones target H3-H4 and H2A-H2B, respectively. In conclusion, our results identify BKRF4 as a histone chaperone encoded by the Epstein-Barr virus, representing a typical histone chaperone found in a non-eukaryote. We envision that more histone chaperones await identification and characterization in DNA viruses and even archaea.

Regulation of pancreatic KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported KATP channel cryo-EM structures in the presence and absence of pharmacological inhibitors and ATP, focusing on the mechanisms by which inhibitors act as pharmacological chaperones of KATP channels (Martin et al., 2019). Here we analyzed the same cryo-EM datasets with a focus on channel conformational dynamics to elucidate structural correlates pertinent to ligand interactions and channel gating. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain, while depleting one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformational change remodels a network of intra- and inter-subunit interactions as well as the ATP and PIP2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1's ABC module involving residues implicated in channel function and showed a SUR1 residue, K134, participates in PIP2 binding. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP2 binding, suggesting a mechanism for competitive gating by ATP and PIP2.

Linker histone H1, facilitated by its chaperones, plays an essential role in regulating gene expression by maintaining chromatin's higher-order structure and epigenetic state. However, we know little about the structural mechanism of how the chaperones recognize linker histones and conduct their function. Here, we used biophysical and biochemical methods to investigate the recognition of human linker histone isoform H1.10 by the TAF-Iβ chaperone. Both H1.10 and TAF-Iβ proteins consist of folded cores and disordered tails. We found that H1.10 formed a complex with TAF-Iβ in a 2:2 stoichiometry. Using distance restraints obtained from methyl-TROSY NMR and spin labels, we built a structural model for the core region of the complex. In the model, the TAF-Iβ core interacts with the globular domain of H1.10 mainly through electrostatic interactions. We confirmed the interactions by measuring the effects of mutations on the binding affinity. A comparison of our structural model with the chromatosome structure shows that TAF-Iβ blocks the DNA binding sites of H1.10. Our study provides insights into the structural mechanism whereby TAF-Iβ functions as a chaperone by preventing H1.10 from interacting with DNA directly.

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. The mechanism of how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs, with a compact core structure, exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL-Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.

Predicting the various binding sites of a protein from its structure sheds light on its function and paves the way towards design of interaction inhibitors. Here, we report ScanNet, a freely available web server for prediction of protein-protein, protein - disordered protein and protein - antibody binding sites from structure. ScanNet (Spatio-Chemical Arrangement of Neighbors Network) is an end-to-end, interpretable geometric deep learning model that learns spatio-chemical patterns directly from 3D structures. ScanNet consistently outperforms Machine Learning models based on handcrafted features and comparative modeling approaches. The web server is linked to both the PDB and AlphaFoldDB, and supports user-provided structure files. Predictions can be readily visualized on the website via the Molstar web app and locally via ChimeraX. ScanNet is available at http://bioinfo3d.cs.tau.ac.il/ScanNet/.

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

Human RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via interactions with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the MED25 transactivator binding ACID domain on the one hand, and the C-terminal α3 helix of NS1, with an additional contribution of the globular domain of NS1, on the other hand. By NMR we show that the NS1 α3 sequence primarily binds to the MED25 ACID H2 face, similarly to the α-helical transactivation domains (TADs) of transcription regulators such as Herpex simplex VP16 and ATF6α, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 could compete with ATF6α TAD for binding to MED25. These findings point to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II.

Interfaces of contact between proteins play important roles in determining the proper structure and function of protein-protein interactions (PPIs). Therefore, to fully understand PPIs, we need to better understand the evolutionary design principles of PPI interfaces. Previous studies have uncovered that interfacial sites are more evolutionarily conserved than other surface protein sites. Yet, little is known about the nature and relative importance of evolutionary constraints in PPI interfaces. Here, we explore constraints imposed by the structure of the microenvironment surrounding interfacial residues on residue evolutionary rate using a large dataset of over 700 structural models of baker's yeast PPIs. We find that interfacial residues are, on average, systematically more conserved than all other residues with a similar degree of total burial as measured by relative solvent accessibility (RSA). Besides, we find that RSA of the residue when the PPI is formed is a better predictor of interfacial residue evolutionary rate than RSA in the monomer state. Furthermore, we investigate four structure-based measures of residue interfacial involvement, including change in RSA upon binding (ΔRSA), number of residue-residue contacts across the interface, and distance from the center or the periphery of the interface. Integrated modeling for evolutionary rate prediction in interfaces shows that ΔRSA plays a dominant role among the four measures of interfacial involvement, with minor, but independent contributions from other measures. These results yield insight into the evolutionary design of interfaces, improving our understanding of the role that structure plays in the molecular evolution of PPIs at the residue level.

Capsid assembly pathways are strongly conserved in the complex dsDNA viruses, where major capsid proteins (MCP) self-assemble into icosahedral procapsid shells, chaperoned by a scaffolding protein. Without a scaffold, the capsid proteins aggregate and form aberrant structures. This, coupled with the rapid co-polymerization of MCP and scaffolding proteins, has thwarted characterization of the earliest steps in shell assembly. Here we interrogate the structure and biophysical properties of a soluble, assembly-deficient phage lambda major capsid protein, MCP(W308A). The mutant protein is folded, soluble to high concentrations and binds to the scaffolding protein in an apparent SP2:MCP(W308A)1 stoichiometry but does not assemble beyond this initiating complex. The MCP(W308A) crystal structure was solved to 2.7 Å revealing the canonical HK97 fold in a "pre-assembly" conformation featuring the conserved N-arm and E-loops folded into the body of the protein. Structural, biophysical and computational analyses suggest that MCP(W308A) is thermodynamically trapped in this pre-assembly conformation precluding self-association interactions required for shell assembly. A model is described wherein dynamic interactions between MCP proteins play an essential role in high fidelity viral shell assembly. Scaffold-chaperoned MCP polymerization is a strongly conserved process in all the large dsDNA viruses and our results provide insight into this primordial complex in solution and have broad biological significance in our understanding of virus assembly mechanisms.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Human immunodeficiency virus (HIV) Gag drives virus particle assembly. The capsid (CA) domain is critical for Gag multimerization mediated by protein-protein interactions. The Gag protein interaction network defines critical aspects of the retroviral lifecycle at steps such as particle assembly and maturation. Previous studies have demonstrated that the immature particle morphology of HIV-2 is intriguingly distinct relative to that of HIV-1. Based upon this observation, we sought to determine the amino acid residues important for virus assembly that might help explain the differences between HIV-1 and HIV-2. To do this, we conducted site-directed mutagenesis of targeted locations in the HIV-2 CA domain of Gag and analyzed various aspects of virus particle assembly. A panel of 31 site-directed mutants of residues that reside at the HIV-2 CA inter-hexamer interface, intra-hexamer interface and CA inter-domain linker were created and analyzed for their effects on the efficiency of particle production, particle morphology, particle infectivity, Gag subcellular distribution and in vitro protein assembly. Seven conserved residues between HIV-1 and HIV-2 (L19, A41, I152, K153, K157, N194, D196) and two non-conserved residues (G38, N127) were found to significantly impact Gag multimerization and particle assembly. Taken together, these observations complement structural analyses of immature HIV-2 particle morphology and Gag lattice organization as well as provide important comparative insights into the key amino acid residues that can help explain the observed differences between HIV immature particle morphology and its association with virus replication and particle infectivity.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

Alzheimer's disease (AD) hallmarks include the aggregation of amyloid-β (Aβ), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aβ42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aβ42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aβ client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aβ42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aβ42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

A characteristic hallmark of Alzheimer's Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aβ plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 - 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297-391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.

Identification of genes that reliably mark distinct cell types is key to leveraging single-cell RNA sequencing to better understand organismal biology. Such genes are usually chosen by measurement of differential expression between groups of cells and selecting those with the greatest magnitude or most statistically significant change. Many methods have been developed for performing such analyses, but no single, best method has emerged. Validating the results of these analyses is costly in terms of time, effort and resources. We demonstrate that applying an ensemble of such methods robustly identifies genes that mark cells that cluster together and that show restricted expression assessed by antisense mRNA in situ and immunofluorescence. This technique is easily extensible to any number of differential expression methods and the inclusion of additional methods is expected to result in further improvement in performance.

Magnesium ions (Mg2+) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg2+ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg2+ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg2+ channels and transporters. In this review article, we provide an overview of the families of Mg2+ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.