The starting point of this work was a SARS-CoV-2 neutralizing peptide (LW25.13), which binds to the receptor-binding domain of the viral spike protein and inhibits the attachment of the virus to its cellular receptor ACE2. As LW25.13 is unable to neutralize later SARS-CoV-2 variants, such as omicron, we have extended the neutralization breadth of LW25.13 through structural and bioinformatic analysis. This involved the systematic variation of a range of positions and yielded peptides neutralizing SARS-CoV-2 beta and omicron at low nanomolar concentrations, while preserving the strong neutralizing capacity against earlier virus variants (wild-type, alpha, delta), as well as the proteolytic stability and α-helical conformation of the peptide. This gain in neutralizing breadth illustrates the utility of the peptide as a scaffold that can be adapted to different virus variants, which may prove useful for the development of peptides against new coronavirus variants of concern in the future.

Electrostatic interactions between charged amino acid side chains play an important role for the stability of α-helices. The strength of these interactions depends on the protonation state of the residues, which is determined by the pH of the environment. Force fields used for molecular dynamics (MD) simulations of such systems should therefore accurately model charged interactions and their dependence on the protonation state. We performed MD simulations for two distinct protonation states on a set of helical peptides known to exhibit various degrees of pH-dependence in experiments and compared two recommended combinations of AMBER protein force field and water model: ff14SB + TIP3P and ff19SB + OPC. While both combinations model similar side chain interactions for the helical states of the peptides, they showed some differences in the overall helical content and the stability of the hydrogen bonds. Nevertheless, both combinations were capable of detecting protonation-dependent structural changes in the peptides. This could be useful for identifying pH-sensitive sites in helical peptides or for designing pH-dependent switches.

The display of a repetitive array of antigens on the surface of nanoparticles has been shown to effectively trigger the activation of cognate B cells and to increase humoral immune responses. Building on extensive knowledge from the development and clinical use of virus-like particle (VLP) vaccines against Human Papilloma Viruses (HPV), we developed an HPV-based nanoparticle carrier presenting Human Immunodeficiency Virus Type 1 (HIV-1) envelope glycoprotein (Env) as a heterologous vaccine antigen on the surface. After purification of the L1 capsid protein of HPV-16 from a bacterial expression system and assembly of L1 proteins into VLPs (L1-VLPs), we conjugated HIV-1 Env trimers with the C-terminus to the surface of the nanoparticles (L1-Env) using copper-free click reactions via Dibenzocyclooctyne (DBCO)-terminated linker molecules. These L1-Env nanoparticles activated Env-specific B cells more efficiently than non-conjugated Env in a B cell receptor-dependent manner. In general, immunization of mice with L1-Env nanoparticles resulted in Env- and L1-specific antibody responses without the need for an adjuvant. L1-Env nanoparticles induced significantly higher Env-specific antibody responses in comparison to a control group that received a mixture of Env trimers and uncoupled L1-VLPs. Concurrently, we observed decreased L1-specific antibody responses in comparison to mice that were immunized with uncoupled L1-VLPs suggesting partial shielding of L1 epitopes by conjugated Env trimers. In summary, HPV-based nanoparticles provide an attractive novel approach for combinatorial vaccines against sexually transmitted pathogens.

Antibody mimetic peptides have evolved as versatile tools for biomedical applications, based on their ability to interfere with protein-protein interactions. We had previously designed a functional mimic of the broadly neutralizing HIV-1 antibody b12 that recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120. The molecular details of the interaction of a linear variant of this peptide (H1H3s) with gp120 have now been characterized through cross-linking mass spectrometry, confirming the proposed involvement of the CD4 binding site of gp120 in the interaction. In addition, a variant of the b12 mimetic peptide composed mostly of D-amino acids was shown to be stable towards proteolytic degradation, while the binding and HIV-1 neutralizing properties were largely preserved. Furthermore, a peptide variant in which aspartate residues were replaced with lysine was shown to strongly enhance infection of cells with HIV-1 and GALV glycoprotein pseudotyped viral vectors, respectively, introducing this peptide as a tool to facilitate retroviral gene transfer. Collectively, the presented results highlight the versatile potential therapeutic and gene transfer applications of H1H3s and its variants in particular, as well as antibody mimetic peptides in general.

IntroductionConformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display.MethodsTo explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner.ResultsMicrospheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation.DiscussionIn comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.

Ensuring site-selectivity in covalent chemical modification of proteins is one of the major challenges in chemical biology and related biomedical disciplines. Most current strategies either utilize the selectivity of proteases, or are based on reactions involving the thiol groups of cysteine residues. We have modified a pair of heterodimeric coiled-coil peptides to enable the selective covalent stabilization of the dimer without using enzymes or cysteine moieties. Fusion of one peptide to the protein of interest, in combination with linking the desired chemical modification to the complementary peptide, facilitates stable, regio-selective attachment of the chemical moiety to the protein, through the formation of the covalently stabilized coiled-coil. This ligation method, which is based on the formation of isoeptide and squaramide bonds, respectively, between the coiled-coil peptides, was successfully used to selectively modify the HIV-1 envelope glycoprotein. Covalent stabilization of the coiled-coil also facilitated truncation of the peptides by one heptad sequence. Furthermore, selective addressing of individual positions of the peptides enabled the generation of mutually selective coiled-coils. The established method, termed Bind & Bite, can be expected to be beneficial for a range of biotechnological and biomedical applications, in which chemical moieties need to be stably attached to proteins in a site-selective fashion.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50 value of 3nm for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.

Alzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.

ABSTRACTVaricella zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. Here, the crystal structure of the VZV Orf24-Orf27 complex is described, representing the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled capsids from the nucleus. While previous studies have primarily emphasized the finding that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focusses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. CoIP and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of the NEC formation thermodynamic parameters of three prototypical α-, β- and γ herpesviruses, i.e. VZV, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) reveals highly similar binding affinities for the autologous interaction with some specific differences in the enthalpy and entropy terms. Computational alanine scanning and structural comparisons highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses. Combined, these data allow to explain the distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings might prove highly valuable when attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

The replication of human cytomegalovirus (HCMV) involves a process termed nuclear egress, which enables translocation of newly formed viral capsids from the nucleus into the cytoplasm. The HCMV core nuclear egress complex (core NEC), a heterodimer of viral proteins pUL50 and pUL53, is therefore considered a promising target for new antiviral drugs. We have recently shown that a 29-mer peptide presenting an N-terminal alpha-helical hook-like segment of pUL53, through which pUL53 interacts with pUL50, binds to pUL50 with high affinity, and inhibits the pUL50–pUL53 interaction in vitro. Here, we show that this peptide is also able to interfere with HCMV infection of cells, as well as with core NEC formation in HCMV-infected cells. As the target of the peptide, i.e., the pUL50–pUL53 interaction, is localized at the inner nuclear membrane of the cell, the peptide had to be equipped with translocation moieties that facilitate peptide uptake into the cell and the nucleus, respectively. For the resulting fusion peptide (NLS-CPP-Hook), specific cellular and nuclear uptake into HFF cells, as well as inhibition of infection with HCMV, could be demonstrated, further substantiating the HCMV core NEC as a potential antiviral target.

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.

Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural–functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50–pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

Protein stability limitations often hamper the exploration of proteins as drug targets. Here, we show that the application of PROSS server algorithms to the ligand-binding domain of human estrogen receptor alpha (hERα) enabled the development of variant ERPRS* that comprises 24 amino acid substitutions and exhibits multiple improved characteristics. The protein displays enhanced production rates in E. coli, crystallizes readily and its thermal stability is increased significantly by 23 °C. hERα is a nuclear receptor (NR) family member. In NRs, protein function is allosterically regulated by its interplay with small molecule effectors and the interaction with coregulatory proteins. The in-depth characterization of ERPRS* shows that these cooperative effects are fully preserved despite that 10% of all residues were substituted. Crystal structures reveal several salient features, i.e. the introduction of a tyrosine corner in a helix-loop-helix segment and the formation of a novel surface salt bridge network possibly explaining the enhanced thermal stability. ERPRS* shows that prior successes in computational approaches for stabilizing proteins can be extended to proteins with complex allosteric regulatory behaviors as present in NRs. Since NRs including hERα are implicated in multiple diseases, our ERPRS* variant shows significant promise for facilitating the development of novel hERα modulators.