Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavH.

L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.

Actin Bundles Dynamics and Architecture.

Cells use the actin cytoskeleton for many of their functions, including their division, adhesion, mechanosensing, endo- and phagocytosis, migration, and invasion. Actin bundles are the main constituent of actin-rich structures involved in these processes. An ever-increasing number of proteins that crosslink actin into bundles or regulate their morphology is being identified in cells. With recent advances in high-resolution microscopy and imaging techniques, the complex process of bundles formation and the multiple forms of physiological bundles are beginning to be better understood. Here, we review the physiochemical and biological properties of four families of highly conserved and abundant actin-bundling proteins, namely, α-actinin, fimbrin/plastin, fascin, and espin. We describe the similarities and differences between these proteins, their role in the formation of physiological actin bundles, and their properties-both related and unrelated to their bundling abilities. We also review some aspects of the general mechanism of actin bundles formation, which are known from the available information on the activity of the key actin partners involved in this process.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes strictly relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a unique mode in a 1:2 stoichiometry with picomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the grove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved via a combination of fast association via the core and very slow dissociation dictated by the arm. Similarly to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by ADF/cofilin, which correlated with the increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Pointed-end processive elongation of actin filaments by Vibrio effectors VopF and VopL.

According to the cellular actin dynamics paradigm, filaments grow at their barbed ends and depolymerize predominantly from their pointed ends to form polar structures and do productive work. We show that actin can elongate at the pointed end when assisted by Vibrio VopF/L toxins, which act as processive polymerases. In cells, processively moving VopF/L speckles are inhibited by factors blocking the pointed but not barbed ends. Multispectral single-molecule imaging confirmed that VopF molecules associate with the pointed end, actively promoting its elongation even in the presence of profilin. Consequently, VopF/L can break the actin cytoskeleton's polarity by compromising actin-based cellular processes. Therefore, actin filament design allows processive growth at both ends, which suggests unforeseen possibilities for cellular actin organization, particularly in specialized cells and compartments.

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity.

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-ß4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.

Allosteric regulation controls actin-bundling properties of human plastins.

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis and other cellular processes by organizing strikingly different actin assemblies as in aligned bundles and branched networks. We propose that this ability of human plastins stems from an allosteric communication between their actin-binding domains (ABD1/2) engaged in a tight spatial association. Here we show that ABD2 can bind actin three orders of magnitude stronger than ABD1, unless the domains are involved in an equally strong inhibitory engagement. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface greatly weakened their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge and domain separation in parallel bundles. We predict that a strong and tunable allosteric inhibition between the domains allows plastins to modulate the cross-linking strength, contributing to remodeling of actin assemblies of different morphologies defining the unique place of plastins in actin organization.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode.

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 pandemic. The emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that can inhibit the virus. One component of the innate immune system with broad antipathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The ability of defensins to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of proteins with high conformational plasticity. We found that human neutrophil α-defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity that is more than 20 fold stronger than its binding to serum albumin. As such, HNP1, as well as a θ-defensin retrocyclin RC-101, both interfere with Spike-mediated membrane fusion, Spike-pseudotyped lentivirus infection, and authentic SARS-CoV-2 infection in cell culture. These effects correlate with the abilities of the defensins to destabilize and precipitate Spike protein and inhibit the interaction of Spike with the ACE2 receptor. Serum reduces the anti-SARS-CoV-2 activity of HNP1, though at high concentrations, HNP1 was able to inactivate the virus even in the presence of serum. Overall, our results suggest that defensins can negatively affect the native conformation of SARS-CoV-2 Spike, and that α- and θ-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Opposing activities of IFITM proteins in SARS-CoV-2 infection.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain- and loss-of-function approaches. Mechanistically, SARS-CoV-2 restriction occurred independently of IFITM3 S-palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis-promoting YxxФ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal SARS-CoV-2 restriction.

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts.

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Opposing activities of IFITM proteins in SARS-CoV-2 infection.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. Here we show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by expression of human IFITM1, IFITM2, and IFITM3, using both gain- and loss-of-function approaches. Mechanistically, restriction of SARS-CoV-2 occurred independently of IFITM3 S -palmitoylation sites, indicating a restrictive capacity that is distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the human IFITM3 endocytosis-promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which reportedly increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into strong enhancers of infection. In sum, these data uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal virus restriction. Indeed, the net effect of IFITM3 on SARS-CoV-2 infections may be a result of these opposing activities, suggesting that shifts in the balance of these activities could be coopted by viruses to escape this important first line innate defense mechanism.

Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.

Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent osteoporosis are understood. Of the over 20 identified osteoporosis-linked PLS3 mutations, we investigated all five that are expected to produce full-length protein. One of the mutations distorted an actin-binding loop in the second actin-binding domain of PLS3 and abolished F-actin bundling as revealed by cryo-EM reconstruction and protein interaction assays. Surprisingly, the remaining four mutants fully retained F-actin bundling ability. However, they displayed defects in Ca2+ sensitivity: two of the mutants lost the ability to be inhibited by Ca2+, while the other two became hypersensitive to Ca2+. Each group of the mutants with similar biochemical properties showed highly characteristic cellular behavior. Wild-type PLS3 was distributed between lamellipodia and focal adhesions. In striking contrast, the Ca2+-hyposensitive mutants were not found at the leading edge but localized exclusively at focal adhesions/stress fibers, which displayed reinforced morphology. Consistently, the Ca2+-hypersensitive PLS3 mutants were restricted to lamellipodia, while chelation of Ca2+ caused their redistribution to focal adhesions. Finally, the bundling-deficient mutant failed to co-localize with any F-actin structures in cells despite a preserved F-actin binding through a non-mutation-bearing actin-binding domain. Our findings revealed that severe osteoporosis can be caused by a mutational disruption of the Ca2+-controlled PLS3's cycling between adhesion complexes and the leading edge. Integration of the structural, biochemical, and cell biology insights enabled us to propose a molecular mechanism of plastin activity regulation by Ca2+.

Plastin 3 in X-Linked Osteoporosis: Imbalance of Ca2+-Dependent Regulation Is Equivalent to Protein Loss.

Osteogenesis imperfecta is a genetic disorder disrupting bone development and remodeling. The primary causes of osteogenesis imperfecta are pathogenic variants of collagen and collagen processing genes. However, recently variants of the actin bundling protein plastin 3 have been identified as another source of osteogenesis imperfecta. Plastin 3 is a highly conserved protein involved in several important cellular structures and processes and is controlled by intracellular Ca2+ which potently inhibits its actin-bundling activity. The precise mechanisms by which plastin 3 causes osteogenesis imperfecta remain unclear, but recent advances have contributed to our understanding of bone development and the actin cytoskeleton. Here, we review the link between plastin 3 and osteogenesis imperfecta highlighting in vitro studies and emphasizing the importance of Ca2+ regulation in the localization and functionality of plastin 3.

Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins.

Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP-G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of "muscle" (CFL2) and "non-muscle" (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins.

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.

The Roles of Actin-Binding Domains 1 and 2 in the Calcium-Dependent Regulation of Actin Filament Bundling by Human Plastins.

The actin cytoskeleton is a complex network controlled by a vast array of intricately regulated actin-binding proteins. Human plastins (PLS1, PLS2, and PLS3) are evolutionary conserved proteins that non-covalently crosslink actin filaments into tight bundles. Through stabilization of such bundles, plastins contribute, in an isoform-specific manner, to the formation of kidney and intestinal microvilli, inner ear stereocilia, immune synapses, endocytic patches, adhesion contacts, and invadosomes of immune and cancer cells. All plastins comprise an N-terminal Ca2+-binding regulatory headpiece domain followed by two actin-binding domains (ABD1 and ABD2). Actin bundling occurs due to simultaneous binding of both ABDs to separate actin filaments. Bundling is negatively regulated by Ca2+, but the mechanism of this inhibition remains unknown. In this study, we found that the bundling abilities of PLS1 and PLS2 were similarly sensitive to Ca2+ (pCa50 ~6.4), whereas PLS3 was less sensitive (pCa50 ~5.9). At the same time, all three isoforms bound to F-actin in a Ca2+-independent manner, suggesting that binding of only one of the ABDs is inhibited by Ca2+. Using limited proteolysis and mass spectrometry, we found that in the presence of Ca2+ the EF-hands of human plastins bound to an immediately adjacent sequence homologous to canonical calmodulin-binding peptides. Furthermore, our data from differential centrifugation, Förster resonance energy transfer, native electrophoresis, and chemical crosslinking suggest that Ca2+ does not affect ABD1 but inhibits the ability of ABD2 to interact with actin. A structural mechanism of signal transmission from Ca2+ to ABD2 through EF-hands remains to be established.

Targeting and inactivation of bacterial toxins by human defensins.

Defensins, as a prominent family of antimicrobial peptides (AMP), are major effectors of the innate immunity with a broad range of immune modulatory and antimicrobial activities. In particular, defensins are the only recognized fast-response molecules that can neutralize a broad range of bacterial toxins, many of which are among the deadliest compounds on the planet. For a decade, the mystery of how a small and structurally conserved group of peptides can neutralize a heterogeneous group of toxins with little to no sequential and structural similarity remained unresolved. Recently, it was found that defensins recognize and target structural plasticity/thermodynamic instability, fundamental physicochemical properties that unite many bacterial toxins and distinguish them from the majority of host proteins. Binding of human defensins promotes local unfolding of the affected toxins, destabilizes their secondary and tertiary structures, increases susceptibility to proteolysis, and leads to their precipitation. While the details of toxin destabilization by defensins remain obscure, here we briefly review properties and activities of bacterial toxins known to be affected by or resilient to defensins, and discuss how recognized features of defensins correlate with the observed inactivation.