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© The Authors Journal compilation © 2014 Biochemical Society.Amyloid fibrils are self-propagating entities that spread pathology in several devastating disorders including Alzheimer's disease (AD). In AD, amyloid-β (Aβ) peptides form extracellular plaques

© 2014 American Chemical Society.The activity of the M2 proton channel of the influenza A virus is controlled by pH. The tautomeric state and conformation of His37, a key residue in the M2 transmembrane four-helix bundle, controls the gating of the channel

Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogue

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter p

Foldamers provide an attractive medium to test the mechanisms by which biological macromolecules fold into complex three-dimensional structures, and ultimately to design novel protein-like architectures with properties unprecedented in nature. Here, we des

Protein designers use a wide variety of software tools for de novo design, yet their repertoire still lacks a fast and interactive all-atom search engine. To solve this, we have built the Suns program: a real-time, atomic search engine integrated into the

Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibod

The pH-regulated M2 proton channel from the influenza A virus has a His-tetrad in its transmembrane (TM) domain that is essential for proton conduction. At neutral pH, the tetrad has been observed in two distinct configurations, the "His-box" and "dimer-of

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A

We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production) system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple mea

The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the

Generating stimulus-responsive, allosteric signaling de novo is a significant challenge in protein design. In natural systems like bacterial histidine kinases (HKs), signal transduction occurs when ligand binding initiates a signal that is amplified across biological membranes over long distances to induce large-scale rearrangements and phosphorylation relays. Here, we ask whether our understanding of protein design and multi-domain, intramolecular signaling has progressed sufficiently to enable engineering of a HK with tunable de novo components. We generated de novo metal-binding sensor domains and substituted them for the native sensor domain of a transmembrane HK, affording chimeras that transduce signals initiated from a de novo sensor. Signaling depended on the designed sensor's stability and the interdomain linker's phase and length. These results show the usefulness of de novo design to elucidate biochemical mechanisms and principles for design of new signaling systems.

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein-protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO 2 F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery., Competing Interests: Competing Interests The authors have no competing interests to disclose.

Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film., Competing Interests: Declaration of interests Z.J.G. and C.S.M. hold patents related to the MULTI-seq barcoding method., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies., (© 2024. The Author(s).)

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

Insoluble amyloids rich in cross-β fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloids in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aβ40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) poses a major challenge to vaccines and antiviral therapeutics due to their extensive evasion of immunity. Aiming to develop potent and broad-spectrum anticoronavirus inhibitors, we generated A1-(GGGGS)7-HR2m (A1L35HR2m) by introducing an angiotensin-converting enzyme 2 (ACE2)-derived peptide A1 to the N terminus of the viral HR2-derived peptide HR2m through a long flexible linker, which showed significantly improved antiviral activity. Further cholesterol (Chol) modification at the C terminus of A1L35HR2m greatly enhanced the inhibitory activities against SARS-CoV-2, SARS-CoV-2 VOCs, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses, with IC 50 values ranging from 0.16 to 5.53 nM. A1L35HR2m-Chol also potently inhibits spike-protein-mediated cell-cell fusion and the replication of authentic Omicron BA.2.12.1, BA.5, and EG.5.1. Importantly, A1L35HR2m-Chol distributed widely in respiratory tract tissue and had a long half-life (>10 h) in vivo. Intranasal administration of A1L35HR2m-Chol to K18-hACE2 transgenic mice potently inhibited Omicron BA.5 and EG.5.1 infection both prophylactically and therapeutically., Competing Interests: Declaration of interests B.D., W.B., S.Y., K.T., and G.C. are the inventors of a provisional patent filed by Westlake University and The University of Hong Kong., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Discovering ligands for amyloid fibrils, such as those formed by the tau protein, is an area of great current interest. In recent structures, ligands bind in stacks in the tau fibrils to reflect the rotational and translational symmetry of the fibril itself; in these structures, the ligands make few interactions with the protein but interact extensively with each other. To exploit this symmetry and stacking, we developed SymDOCK, a method to dock molecules that follow the protein's symmetry. For each prospective ligand pose, we apply the symmetry operation of the fibril to generate a self-interacting and fibril-interacting stack, checking that doing so will not cause a clash between the original molecule and its image. Absent a clash, we retain that pose and add the ligand-ligand van der Waals energy to the ligand's docking score (here using DOCK3.8). We can check these geometries and energies using an implementation of ANI, a neural-network-based quantum-mechanical evaluation of the ligand stacking energies. In retrospective calculations, symmetry docking can reproduce the poses of three tau PET tracers whose structures have been determined. More convincingly, in a prospective study, SymDOCK predicted the structure of the PET tracer MK-6240 bound in a symmetrical stack to AD PHF tau before that structure was determined; the docked pose was used to determine how MK-6240 fit the cryo-EM density. In proof-of-concept studies, SymDOCK enriched known ligands over property-matched decoys in retrospective screens without sacrificing docking speed and can address large library screens that seek new symmetrical stackers. Future applications of this approach will be considered.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Selective proton transport through proteins is essential for forming and using proton gradients in cells. Protons are conducted along hydrogen-bonded 'wires' of water molecules and polar side chains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways, inferred from static protein structures. Here we hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess protons in the water wire. To test this hypothesis, we performed molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 10 6 -fold more selective for H + over Na + . These studies inform the mechanisms of biological proton conduction and the principles for engineering proton-conductive materials., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Accumulation of filamentous aggregates of tau protein in the brain is a pathological hallmark of Alzheimer's disease (AD) and many other neurodegenerative tauopathies. The filaments adopt disease-specific cross-β amyloid conformations that self-propagate and are implicated in neuronal loss. Development of molecular diagnostics and therapeutics is of critical importance. However, mechanisms of small molecule binding to the amyloid core is poorly understood. We used cryo-electron microscopy to determine a 2.7 Å structure of AD patient-derived tau paired-helical filaments bound to the PET ligand GTP-1. The compound is bound stoichiometrically at a single site along an exposed cleft of each protofilament in a stacked arrangement matching the fibril symmetry. Multiscale modeling reveals pi-pi aromatic interactions that pair favorably with the small molecule-protein contacts, supporting high specificity and affinity for the AD tau conformation. This binding mode offers critical insight into designing compounds to target different amyloid folds found across neurodegenerative diseases., (© 2023. The Author(s).)

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections., (© 2023. The Author(s).)

In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains in vitro, as well as amyloid-β (Aβ) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aβ and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.

Studies show that patients with Alzheimer's disease (AD) have both Aβ and tau prions, and thus, AD is a double-prion disease. AD patients with the greatest longevity exhibited low levels of both Aβ and tau prions; tau prions were nearly absent in the brains of almost half of the patients who lived beyond 80 years of age. Using cellular bioassays for prions in postmortem samples, we found that both Aβ and tau proteins misfold into prions leading to AD, which is either a sporadic or familial dementing disorder. Although AD is transmissible experimentally, there is no evidence that AD is either communicable or contagious. Since the progression of AD correlates poorly with insoluble Aβ in the central nervous system (CNS), it was difficult to distinguish between inert amyloids and Aβ prions. To measure the progression of AD, we devised rapid bioassays to measure the abundance of isoform-specific Aβ prions in the brains of transgenic (Tg) mice and in postmortem human CNS samples from AD victims and people who died of other neurodegenerative diseases (NDs). We found significant correlations between the longevity of individuals with AD, sex, and genetic background, despite the fact that all postmortem brain tissue had essentially the same confirmed neuropathology.Although brains from all AD patients had measurable levels of Aβ prions at death, the oldest individuals had lower Aβ prion levels than the younger ones. Additionally, the long-lived individuals had low tau prion levels that correlated with the extent of phosphorylated tau (p-tau). Unexpectedly, a longevity-dependent decrease in tau prions was found in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the tau prion levels decreased exponentially with respect to the age at death with a half-time of approximately one decade, and this correlated with the abundance of phosphorylated tau.Even though our findings with tau prions were not unexpected, they were counterintuitive; thus, tau phosphorylation and tau prion activity decreased exponentially with longevity in patients with AD ranging from ages 37 to 99 years. Our findings demonstrated an inverse correlation between longevity in AD patients and the abundance of neurotoxic tau prions. Moreover, our discovery may have profound implications for the selection of phenotypically distinct patient populations and the development of diagnostics and effective therapeutics for AD., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19 F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19 F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. Here, we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa V ) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3-a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3-histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic 'groove' of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Natural killer (NK) cells can detect antibody-coated cells through recognition by the CD16 Fc receptor. The importance of CD16 in human NK cell biology has long been appreciated, but how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. We reveal that the mouse CD3ζ transmembrane domain adopts a tightly packed confirmation, preventing association with CD16, whereas human CD3ζ adopts a versatile configuration that accommodates receptor assembly., (© 2022 Aguilar et al.)

Brilacidin, a mimetic of host defense peptides (HDPs), is currently in Phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by inactivating the virus. In this study, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on the host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS-CoV-2 pseudovirus into multiple cell lines, and heparin, an HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS-CoV-2 pseudovirus cell entry. In addition, we found that brilacidin has broad-spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, and HCoV-NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on the host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host-targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV-OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad-spectrum antiviral for coronaviruses including SARS-CoV-2., (© 2022 Wiley Periodicals LLC.)

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer's disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-β (Aβ) into plaques, particularly Aβ40 and Aβ42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aβ fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aβ, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aβ40 and Aβ42, even at a high micromolar concentration, while it does bind to fibrillar Aβ42 and Aβ40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aβ, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2-Aβ fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The influenza A M2 channel, a prototype for viroporins, is an acid-activated viroporin that conducts protons across the viral membrane, a critical step in the viral life cycle. Four central His37 residues control channel activation by binding subsequent protons from the viral exterior, which opens the Trp41 gate and allows proton flux to the interior. Asp44 is essential for maintaining the Trp41 gate in a closed state at high pH, resulting in asymmetric conduction. The prevalent D44N mutant disrupts this gate and opens the C-terminal end of the channel, resulting in increased conduction and a loss of this asymmetric conduction. Here, we use extensive Multiscale Reactive Molecular Dynamics (MS-RMD) and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations with an explicit, reactive excess proton to calculate the free energy of proton transport in this M2 mutant and to study the dynamic molecular-level behavior of D44N M2. We find that this mutation significantly lowers the barrier of His37 deprotonation in the activated state and shifts the barrier for entry to the Val27 tetrad. These free energy changes are reflected in structural shifts. Additionally, we show that the increased hydration around the His37 tetrad diminishes the effect of the His37 charge on the channel's water structure, facilitating proton transport and enabling activation from the viral interior. Altogether, this work provides key insight into the fundamental characteristics of PT in WT M2 and how the D44N mutation alters this PT mechanism, and it expands understanding of the role of emergent mutations in viroporins.

Natural metalloproteins perform many functions - ranging from sensing to electron transfer and catalysis - in which the position and property of each ligand and metal, is dictated by protein structure. De novo protein design aims to define an amino acid sequence that encodes a specific structure and function, providing a critical test of the hypothetical inner workings of (metallo)proteins. To date, de novo metalloproteins have used simple, symmetric tertiary structures - uncomplicated by the large size and evolutionary marks of natural proteins - to interrogate structure-function hypotheses. In this Review, we discuss de novo design applications, such as proteins that induce complex, increasingly asymmetric ligand geometries to achieve function, as well as the use of more canonical ligand geometries to achieve stability. De novo design has been used to explore how proteins fine-tune redox potentials and catalyse both oxidative and hydrolytic reactions. With an increased understanding of structure-function relationships, functional proteins including O 2 -dependent oxidases, fast hydrolases, and multi-proton/multi-electron reductases, have been created. In addition, proteins can now be designed using xeno-biological metals or cofactors and principles from inorganic chemistry to derive new-to-nature functions. These results and the advances in computational protein design suggest a bright future for the de novo design of diverse, functional metalloproteins., Competing Interests: Competing interests The authors declare no competing interests.

Amyloid beta (Aβ) is thought to play a critical role in the pathogenesis of Alzheimer's disease (AD). Prion-like Aβ polymorphs, or "strains", can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of Aβ that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and Aβ strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various Aβ and tau species and used conformation-sensitive fluorescent probes to detect differences in Aβ strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of Aβ emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sporadic AD., (© 2021. The Author(s).)