Trigger Conformational Changes Research Articles

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Molecular machine-based nanomaterials: Recent progress and prospects

Molecular machines are chemical molecules (man-made or natural) which display triggered reversible conformational changes by external stimuli like pH and temperature, followed by observable changes. This property of controllable and coherent motion at molecular level can be observed through the synthesis of macroscopic materials from self-assembly of molecular machines. Due to these properties, molecular machine-based nanomaterials display a wide range of unique characteristics and various useful applications in biomedicine, energy, environmental engineering, etc. This paper reviews recent research articles on some potential applications including biosensing, therapeutics (with a focus on drug delivery system) and catalysis based on MMs and their composites to assess the current practicality of the technology. Although most materials are not ready for real-life applications yet and there is a lack of deep understanding of the efficacy, these nanomaterials display a wide range of very useful and unique characteristics and present a brand-new direction for tackling current challenges.

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody

COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity.To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient.This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions.

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M β-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried β-sheet. The two mutations located in this β-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M β-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Identification and Evaluation of Traditional Chinese Medicine Natural Compounds as Potential Myostatin Inhibitors: An In Silico Approach.

Myostatin (MSTN), a negative regulator of muscle mass, is reported to be increased in conditions linked with muscle atrophy, sarcopenia, and other muscle-related diseases. Most pharmacologic approaches that treat muscle disorders are ineffective, emphasizing the emergence of MSTN inhibition. In this study, we used computational screening to uncover natural small bioactive inhibitors from the Traditional Chinese Medicine database (~38,000 compounds) for the MSTN protein. Potential ligands were screened, based on binding affinity (150), physicochemical (53) and ADMET properties (17). We found two hits (ZINC85592908 and ZINC85511481) with high binding affinity and specificity, and their binding patterns with MSTN protein. In addition, molecular dynamic simulations were run on each complex to better understand the interaction mechanism of MSTN with the control (curcumin) and the hit compounds (ZINC85592908 and ZINC85511481). We determined that the hits bind to the active pocket site (Helix region) and trigger conformational changes in the MSTN protein. Since the stability of the ZINC85592908 compound was greater than the MSTN control, we believe that ZINC85592908 has therapeutic potential against the MSTN protein and may hinder downstream singling by inhibiting the MSTN protein and increasing myogenesis in the skeletal muscle tissues.

Substrate-binding loop interactions with pseudouridine trigger conformational changes that promote catalytic efficiency of pseudouridine kinase PUKI

Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5′-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5′-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their Km values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli strain B (EcPUKI) in various ligation states. The structure of EcPUKI was determined to be similar to PUKI from Arabidopsis thaliana, including an α/β core domain and β-stranded small domain, with dimerization occurring via the β-stranded small domain. In a binary complex, we show that Ser30 in the substrate-binding loop of the small domain mediates interactions with the hallmark N1 atom of pseudouridine nucleobase, causing conformational changes in its quaternary structure. Kinetic and fluorescence spectroscopic analyses also showed that the Ser30-mediated interaction is a prerequisite for conformational changes and subsequent catalysis by EcPUKI. Furthermore, S30A mutation or EcPUKI complexed with other nucleosides homologous to pseudouridine but lacking the pseudouridine-specific N1 atom did not induce such conformational changes, demonstrating the catalytic significance of the proposed Ser30-mediated interaction. These analyses provide structural and functional evidence for a pseudouridine-dependent conformational change of EcPUKI and its functional linkage to catalysis.

PH-Dependent Mechanisms of Influenza Infection Mediated by Hemagglutinin.

Influenza hemagglutinin (HA) is a viral membrane bound protein that plays a critical role in the viral life cycle by mediating entry into target cells. HA exploits the lowering of the pH in the endosomal compartment to initiate a series of conformational changes that promote access of the viral genetic material to the cytoplasm, and hence viral replication. In this review we will first discuss what is known about the structural properties of HA as a function of pH. Next, we will discuss the dynamics and intermediate states of HA. We will then discuss the specific residues that are thought to be titrated by the change in pH and possible mechanisms for the pH triggered conformational changes. Finally, we will discuss small molecules that disrupt the pH trigger and thus serve as potential therapeutic strategies to prevent influenza infection.

ATP hydrolysis and nucleotide exit enhance maltose translocation in the MalFGK2E importer

ATP binding cassette (ABC) transporters employ ATP hydrolysis to harness substrate translocation across membranes. The Escherichia coli MalFGK2E maltose importer is an example of a type I ABC importer and a model system for this class of ABC transporters. The MalFGK2E importer is responsible for the intake of malto-oligossacharides in E.coli. Despite being extensively studied, little is known about the effect of ATP hydrolysis and nucleotide exit on substrate transport. In this work, we studied this phenomenon using extensive molecular dynamics simulations (MD) along with potential of mean force calculations of maltose transport across the pore, in the pre-hydrolysis, post-hydrolysis and nucleotide-free states. We concluded that ATP hydrolysis and nucleotide exit trigger conformational changes that result in the decrease of energetic barriers to maltose translocation towards the cytoplasm, with a concomitant increase of the energy barrier in the periplasmic side of the pore, contributing for the irreversibility of the process. We also identified key residues that aid in positioning and orientation of maltose, as well as a novel binding pocket for maltose in MalG. Additionally, ATP hydrolysis leads to conformations similar to the nucleotide-free state. This study shows the contribution of ATP hydrolysis and nucleotide exit in the transport cycle, shedding light on ABC type I importer mechanisms.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH.

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696-10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered.IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

In vitro reconstitution and biochemical characterization of human phospholipid scramblase 3: phospholipid specificity and metal ion binding studies.

Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of scramblases remains still unknown, and the role of scramblases in phospholipid translocation is heavily debated. hPLSCR3 is the only member of scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminophospholipids such as NBD-PE and NBD-PS but not neutral phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the phospholipid translocation by hPLSCR3.

Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase

BackgroundAlkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. MethodsAs a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. ResultsConformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. ConclusionsOur data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. General significanceA general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.

The Yeast Prion [SWI(+)] Abolishes Multicellular Growth by Triggering Conformational Changes of Multiple Regulators Required for Flocculin Gene Expression.

Although transcription factors are prevalent among yeast prion proteins, the role of prion-mediated transcriptional regulation remains elusive. Here, we show that the yeast prion [SWI(+)] abolishes flocculin (FLO) gene expression and results in a complete loss of multicellularity. Further investigation demonstrates that besides Swi1, multiple other proteins essential for FLO expression, including Mss11, Sap30, and Msn1 also undergo conformational changes and become inactivated in [SWI(+)] cells. Moreover, the asparagine-rich region of Mss11 can exist as prion-like aggregates specifically in [SWI(+)] cells, which are SDS resistant, heritable, and curable, but become metastable after separation from [SWI(+)]. Our findings thus reveal a prion-mediated mechanism through which multiple regulators in a biological pathway can be inactivated. In combination with the partial loss-of-function phenotypes of [SWI(+)] cells on non-glucose sugar utilization, our data therefore demonstrate that a prion can influence distinct traits differently through multi-level regulations, providing insights into the biological roles of prions.

Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling.

Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D3 signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAFII 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains.

Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins

Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.

Histone deacetylase inhibitors cause TP53-dependent induction of p21/Waf1 in tumor cells with TP53 mutations

The p21/Waf1 protein is one of the main regulators of cell cycle arrest and one of the best-known transcriptional targets of the TP53 protein. Here, we demonstrated that there is activation of expression of the p21/Waf1 gene when the cells were treated with sodium butyrate (NaBu), which is a natural histone deacetylase inhibitor, and investigated whether this phenomenon depends on the presence of a functionally active TP53 protein. For this purpose, we compared the effect of NaBu treatment of human cell lines with different TP53 mutation profiles, including wild-type TP53, single nucleotide substitutions, and the complete absence of the TP53 gene. NaBu activated the TP53 protein via hyperacetylation at the lysine residue K382, without significant changes in the level of protein expression. Western blotting showed that the addition of NaBu triggers a significant increase in the p21/Waf1 protein level in both TP53 wild-type cells and in cells with single nucleotide substitutions in the central DNA-binding core domain (DBD) of the TP53 protein. At the same time, no p21/Waf1 protein induction was observed in cells with complete deletion of the TP53 gene. However, NaBu was not able to induce p21/Waf1 production when the expression of TP53 was transiently knocked down by the p53 siRNA. Overall, our results suggest that NaBu-dependent induction of p21/Waf1 does require the presence of TP53 protein, but, unexpectedly, it can occur regardless of mutational changes in the domain responsible for the TP53 binding to DNA. One possible explanations is that NaBu increases the level of TP53 acetylation and the modified protein is able to establish a new network of protein–protein interactions or trigger conformational changes affecting the TP53-dependent transcriptional machinery even when its DNA binding ability is impaired.

Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.

αvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion.