A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies.

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Nanobodies to multiple spike variants and inhalation of nanobody-containing aerosols neutralize SARS-CoV-2 in cell culture and hamsters.

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.

N4-hydroxycytidine, the active compound of Molnupiravir, promotes SARS-CoV-2 mutagenesis and escape from a neutralizing nanobody.

N4-hydroxycytidine (NHC), the active compound of the drug Molnupiravir, is incorporated into SARS-CoV-2 RNA, causing false base pairing. The desired result is an "error catastrophe," but this bears the risk of mutated virus progeny. To address this experimentally, we propagated the initial SARS-CoV-2 strain in the presence of NHC. Deep sequencing revealed numerous NHC-induced mutations and host-cell-adapted virus variants. The presence of the neutralizing nanobody Re5D06 selected for immune escape mutations, in particular p.E484K and p.F490S, which are key mutations of the Beta/Gamma and Omicron-XBB strains, respectively. With NHC treatment, nanobody resistance occurred two passages earlier than without. Thus, within the limitations of this purely in vitro study, we conclude that the combined action of Molnupiravir and a spike-neutralizing antagonist leads to the rapid emergence of escape mutants. We propose caution use and supervision when using Molnupiravir, especially when patients are still at risk of spreading virus.

Barrier properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length.

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phase-entry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFPNTR-variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR·FG interactions is key to transport selectivity.

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins.

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

Our muscles are not just for lifting weights. They also keep us alive. For example, our heartbeat is powered by the muscles in the heart wall. Just like other organs in the body, muscles are made up of cells called muscle fibres. Each muscle fibre is divided into many smaller units, or 'sarcomeres', which contain specialised proteins that pull on each other to produce muscle contractions. Although the structure of mature muscles is rather well understood, we know much less about how muscles develop or how they are maintained throughout adult life. Understanding this is especially important in the case of the heart, because its muscle cells are not replaced throughout our lives. Instead, the heart muscle cells we are born with are maintained as we age while working continuously. This means that the proteins within the heart muscle sarcomeres are continuously under mechanical stress and may need to be repaired. How this repair might happen is not well understood. Nanobodies are very small versions of antibodies that recognise and bind to specific protein targets. In biological research, they are used as a tool to observe proteins of interest within cells. This is done by labelling nanobodies, for example, with chemical fluorophores or fluorescent proteins; once labelled, the nanobody binds to its target protein, and scientists can monitor its location and behaviour within the cell. Cells, and even flies, can also be genetically manipulated to produce labelled nanobodies themselves, which has the advantage of visualising the dynamic behaviour of the target protein in the living cell or organism. To better study the proteins in muscle cells, scientists from two different research groups developed a nanobody 'toolbox' that specifically targets sarcomere proteins. First, Loreau et al. made a 'library' of labelled nanobodies targeting different sarcomere proteins in Drosophila melanogaster fruit flies. Second, they used this library of nanobodies to locate several sarcomere proteins in the mature sarcomeres of different fly muscles. Third, using flies that had been genetically altered to produce the labelled nanobodies in their muscle cells, Loreau et al. were able to observe the behaviour of the target proteins in the living muscle. Together, these experiments showed that one protein in Drosophila that is similar to the human sarcomere protein titin has a similar size to the human version, whereas a second Drosophila titin-like protein is shorter and located at a different place in the sarcomere. Both of these proteins work together to stabilise muscle fibres, which is also the role of human titin. The nanobodies generated here are a significant contribution to the tools available to study muscle development and maintenance. Loreau et al. hope that they will help reveal how sarcomere proteins like titin are maintained, especially in the heart, and ultimately how the heart muscle manages to continue working throughout our lives.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

A simple thermodynamic description of phase separation of Nup98 FG domains.

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules but allows facilitated passage of nuclear transport receptors that shuttle cargoes into or out of nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains, including foremost the charge-depleted FG domain of Nup98. We found that Nup98 FG domains show an LCST-type phase separation, and we provide comprehensive and orthogonal experimental datasets for a quantitative description of this behaviour. A derived thermodynamic model correlates saturation concentration with repeat number, temperature, and ionic strength. It allows estimating the enthalpy, entropy, and ΔG (0.2 kJ/mol, 0.1 kB·T) contributions per repeat to phase separation and inter-repeat cohesion. While changing the cohesion strength strongly impacts the strictness of barrier, these numbers provide boundary conditions for in-depth modelling not only of barrier assembly but also of NPC passage.

Crystal structures of FNIP/FGxxFN motif-containing leucine-rich repeat proteins.

The Cafeteria roenbergensis virus (Crov), Dictyostelium, and other species encode a large family of leucine-rich repeat (LRR) proteins with FGxxFN motifs. We determined the structures of two of them and observed several unique structural features that set them aside from previously characterized LRR family members. Crov588 comprises 25 regular repeats with a LxxLxFGxxFNQxIxENVLPxx consensus, forming a unique closed circular repeat structure. Novel features include a repositioning of a conserved asparagine at the middle of the repeat, a double phenylalanine spine that generates an alternate core packing arrangement, and a histidine/tyrosine ladder on the concave surface. Crov539 is smaller, comprising 12 repeats of a similar LxxLxFGxxFNQPIExVxW/LPxx consensus and forming an unusual cap-swapped dimer structure. The phenylalanine spine of Crov539 is supplemented with a tryptophan spine, while a hydrophobic isoleucine-rich patch is found on the central concave surface. We present a detailed analysis of the structures of Crov588 and Crov539 and compare them to related repeat proteins and other LRR classes.

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic.

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1 * 105 to 1 * 10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1 * 10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1 * 102 TCID50 as the optimal dose for experimental infections. This compelling high susceptibility leading to productive infections in Golden Syrian hamsters must be considered as a potential source of SARS-CoV-2 infection for humans that come into close contact with pet hamsters.

An In Vivo Model for Elucidating the Role of an Erythroid-Specific Isoform of Nuclear Export Protein Exportin 7 (Xpo7) in Murine Erythropoiesis.

Erythroid nuclear condensation is a complex process in which compaction to one-tenth its original size occurs in an active nucleus simultaneously undergoing transcription and cell division. We previously found that the nuclear exportin Exportin7 (Xpo7), which is erythroid- specific and highly induced during terminal erythropoiesis, facilitates nuclear condensation. We also identified a previously unannotated, erythroid-specific isoform of Xpo7 (Xpo7B) containing a novel first exon Xpo7-1b expressed only in late Ter119+ erythroblasts. To better understand the functional difference between the erythroid Xpo7B isoform and the ubiquitous isoform (Xpo7A) containing the original first exon Xpo7-1a, we created gene-targeted mouse models lacking either exon Xpo7-1a or Xpo7-1b, or both exons 4 and 5, which are completely null for Xpo7 expression. We found that deficiency in Xpo7A does not affect steady-state nor stress erythropoiesis. In contrast, mice lacking the erythroid isoform, Xpo7B, exhibit a mild anemia as well as altered stress erythropoiesis. Complete Xpo7 deficiency resulted in partially penetrant embryonic lethality at the stage when definitive erythropoiesis is prominent in the fetal liver. Inducible complete knockdown of Xpo7 confirms that both steady-state erythropoiesis and stress erythropoiesis are affected. We also observe that Xpo7 deficiency downregulates the expression of important stress response factors, such as Gdf15 and Smad3. We conclude that the erythroid-specific isoform of Xpo7 is important for both steady-state and stress erythropoiesis in mice.

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication.

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

Atomic resolution dynamics of cohesive interactions in phase-separated Nup98 FG domains.

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy.

Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP.

Mechanical forces regulate multiple essential pathways in the cell. The nuclear translocation of mechanoresponsive transcriptional regulators is an essential step for mechanotransduction. However, how mechanical forces regulate the nuclear import process is not understood. Here, we identify a highly mechanoresponsive nuclear transport receptor (NTR), Importin-7 (Imp7), that drives the nuclear import of YAP, a key regulator of mechanotransduction pathways. Unexpectedly, YAP governs the mechanoresponse of Imp7 by forming a YAP/Imp7 complex that responds to mechanical cues through the Hippo kinases MST1/2. Furthermore, YAP behaves as a dominant cargo of Imp7, restricting the Imp7 binding and the nuclear translocation of other Imp7 cargoes such as Smad3 and Erk2. Thus, the nuclear import process is an additional regulatory layer indirectly regulated by mechanical cues, which activate a preferential Imp7 cargo, YAP, which competes out other cargoes, resulting in signaling crosstalk.

Therapeutische Nanobodies gegen SARS-CoV-2.

Monoclonal immunoglobulins are widely successful as therapeutics and have also been effective in treating COVID-19. However, their production in mammalian cells is expensive and cannot be scaled to meet the demand in a global pandemic. Camelid VHH antibodies (also called nanobodies), however, can be manufactured cost-efficiently in bacteria or yeast. Here we highlight our progress in developing nanobodies that effectively neutralize SARS-CoV-2 and its variants.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide.

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with 'DNA-specific' DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.

The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models.

The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 µM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) and ~100-fold reductions in Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 100 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.

The copper(II)-binding tripeptide GHK, a valuable crystallization and phasing tag for macromolecular crystallography.

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins.

We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane-sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine-glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.