Trimeric Protein Research Articles

A highly efficient blocking ELISA based on p72 monoclonal antibody for the detection of African swine fever virus antibodies and identification of its linear B cell epitope

Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies.

Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2089.

Zeolitic imidazolate frameworks activate endosomal Toll-like receptors and potentiate immunogenicity of SARS-CoV-2 spike protein trimer.

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework-8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.

Quantitative analysis of MBW complex formation in the context of trichome patterning.

Trichome patterning in Arabidopsis is regulated by R2R3MYB, bHLH and WDR (MBW) genes. These are considered to form a trimeric MBW protein complex that promotes trichome formation. The MBW proteins are engaged in a regulatory network to select trichome cells among epidermal cells through R3MYB proteins that can move between cells and repress the MBW complex by competitive binding with the R2R3MYB to the bHLHL protein. We use quantitative pull-down assays to determine the relative dissociation constants for the protein-protein interactions of the involved genes. We find similar binding strength between the trichome promoting genes and weaker binding of the R3MYB inhibitors. We used the dissociation constants to calculate the relative percentage of all possible complex combinations and found surprisingly low fractions of those complexes that are typically considered to be relevant for the regulation events. Finally, we predict an increased robustness in patterning as a consequence of higher ordered complexes mediated by GL3 dimerization.

Cytoskeletal β-tubulin and cysteine cathepsin L deregulation by SARS-CoV-2 spike protein interaction with the neuronal model cell line SH-SY5Y

SARS-CoV-2 mainly infects the respiratory tract but can also target other organs, including the central nervous system. While it was recently shown that cells of the blood-brain-barrier are permissive to SARS-CoV-2 infection in vitro, it remains debated whether neurons can be infected. In this study, we demonstrate that vesicular stomatitis virus particles pseudotyped with the spike protein of SARS-CoV-2 variants WT, Alpha, Delta and Omicron enter the neuronal model cell line SH-SY5Y. Cell biological analyses of the pseudo-virus treated cultures showed marked alterations in microtubules of SH-SY5Y cells. Because the changes in β-tubulin occurred in most cells, but only few were infected, we further asked whether interaction of the cells with spike protein might be sufficient to cause molecular and structural changes. For this, SH-SY5Y cells were incubated with trimeric spike proteins for time intervals of up to 24 h. CellProfiler™-based image analyses revealed changes in the intensities of microtubule staining in spike protein-incubated cells. Furthermore, expression of the spike protein-processing protease cathepsin L was found to be up-regulated by wild type, Alpha and Delta spike protein pseudotypes and cathepsin L was found to be secreted from spike protein-treated cells. We conclude that the mere interaction of the SARS-CoV-2 with neuronal cells can affect cellular architecture and proteolytic capacities. The molecular mechanisms underlying SARS-CoV-2 spike protein induced cytoskeletal changes in neuronal cells remain elusive and require future studies.

Polyvalent Nanobody Structure Designed for Boosting SARS-CoV-2 Inhibition.

Coronavirus transmission and mutations have brought intensive challenges on pandemic control and disease treatment. Developing robust and versatile antiviral drugs for viral neutralization is highly desired. Here, we created a new polyvalent nanobody (Nb) structure that shows the effective inhibition of SARS-CoV-2 infections. Our polyvalent Nb structure, called "PNS", is achieved by first conjugating single-stranded DNA (ssDNA) and the receptor-binding domain (RBD)-targeting Nb with retained binding ability to SARS-CoV-2 spike protein and then coalescing the ssDNA-Nb conjugates around a gold nanoparticle (AuNP) via DNA hybridization with a desired Nb density that offers spatial pattern-matching with that of the Nb binding sites on the trimeric spike. The surface plasmon resonance (SPR) assays show that the PNS binds the SARS-CoV-2 trimeric spike proteins with a ∼1000-fold improvement in affinity than that of monomeric Nbs. Furthermore, our viral entry inhibition assays using the PNS against SARS-CoV-2 WA/2020 and two recent variants of interest (BQ1.1 and XBB) show an over 400-fold enhancement in viral inhibition compared to free Nbs. Our PNS strategy built on a new DNA-protein conjugation chemistry provides a facile approach to developing robust virus inhibitors by using a corresponding virus-targeting Nb with a desired Nb density.

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial β-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls.Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

Structural dynamics of influenza A (H1N1) hemagglutinin protein: a comparative study of Indian (2018) isolate with its evolutionary neighbor, Californian (2009) strain

This work highlights the structure and dynamics of two trimeric HA proteins of the H1N1 virus from different origins, the pandemic Californian (HACal) and its closest Indian neighbor (HAInd), reported in 2009 and 2018, respectively. Because of mutation, HAInd acquires new N-glycosylation and epitope binding sites along with mutations at RBD, which might trigger an altered viral-host interaction mechanism. Molecular dynamics simulations performed on HA trimers for a period of 250 ns reveal the highly dynamic nature of HACal trimers inherited by the flexibility of HA monomers. In the trimer, the dynamics of one monomer are more pronounced compared to others, and the enhanced dynamics of RBD especially gain attention as they plays a key role during fusion. Conversely, the mutant HAInd trimer effectively establishes more H-bond interactions, and accordingly, the trimer undergoes more stabilized dynamics with a relatively lower amplitude of RBD dynamics, as endorsed by the reduced RMSD, Rg, and SASA variations. The cooperative and anti-cooperative motions dissected for the subdomains of both strains also reveal a prominent anticorrelative motion of RBD against other subdomains. In agreement, the free energy landscape of stable HAInd is also characterized by a single lowest wide energy basin instead of the two minimum energy basins of the HACal trimer. In essence, the mutant HAInd acquires a highly stable conformation with novel functional features, which calls for (i) further investigation on the emerging mutation-mediated variation in viral-host binding mechanism and (ii) the need for further design of site-specific potential inhibitors to face future challenges. Communicated by Ramaswamy H. Sarma

Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

Het-B allorecognition in Podospora anserina is determined by pseudo-allelic interaction of genes encoding a HET and lectin fold domain protein and a PII-like protein.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species.

Oriented display of HIV-1 Env trimers by a novel coupling strategy enhances B cell activation and phagocytosis.

Conformationally 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. To 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. Microspheres 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. In 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.

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling

Key messageRetromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling.In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes.

The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

Structure of human GPR119-Gs complex binding APD597 and characterization of GPR119 binding agonists.

The rhodopsin-like receptor GPR119 plays a crucial role in glucose homeostasis and is an emerging target for the treatment of type 2 diabetes mellitus. In this study, we analyzed the structure of GPR119 with the agonist APD597 bound and in complex with the downstream G protein trimer by single particle cryo-electron microscopy (cryo-EM). Structural comparison in combination with function assay revealed the conservative and specific effects of different kinds of GPR119 agonists. The activation mechanism of GPR119 was analyzed by comparing the conformational changes between the inactive and active states. The interaction between APD597 derivatives and synthetic agonists with GPR119 was analyzed by molecular docking technique, and the necessary structural framework was obtained. The above conclusions can provide structural and theoretical basis for the development of therapeutic drugs for type 2 diabetes mellitus.

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2′ acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.

Allosteric regulation in SARS-CoV-2 spike protein.

Allosteric regulation is common in protein-protein interactions and is thus promising in drug design. Previous experimental and simulation work supported the presence of allosteric regulation in the SARS-CoV-2 spike protein. Here the route of allosteric regulation in SARS-CoV-2 spike protein is examined by all-atom explicit solvent molecular dynamics simulations, contrastive machine learning, and the Ohm approach. It was found that peptide binding to the polybasic cleavage sites, especially the one at the first subunit of the trimeric spike protein, activates the fluctuation of the spike protein's backbone, which eventually propagates to the receptor-binding domain on the third subunit that binds to ACE2. Remarkably, the allosteric regulation routes starting from the polybasic cleavage sites share a high fraction (39-67%) of the critical amino acids with the routes starting from the nitrogen-terminal domains, suggesting the presence of an allosteric regulation network in the spike protein. Our study paves the way for the rational design of allosteric antibody inhibitors.

Strategic design of GalNAc-helical peptide ligands for efficient liver targeting.

There is a growing need for liver-selective drug delivery systems (DDS) in the treatment and diagnosis of liver diseases. The asialoglycoprotein receptor, a trimeric protein specifically expressed in the liver, is a key target for DDS. We hypothesized that peptides with reduced main-chain flexibility and strategically positioned N-acetylgalactosamine (GalNAc) moieties could enhance liver selectivity and uptake efficiency. The helical peptides designed in this study demonstrated superior uptake efficiency and liver selectivity compared with the conventional triantennary GalNAc DDS. These peptides also showed potential in protein delivery. Furthermore, we explored their application in lysosome-targeting chimeras (LYTACs), gaining valuable insights into the requirements for effective LYTAC functionality. This study not only highlights the potential of helical peptides as liver-selective DDS ligands, but also opens avenues for their use in various therapeutic and diagnostic applications, making significant strides in the targeted treatment of liver diseases.

Photosystem I complexes form remarkably stable self-assembled tunneling junctions.

This paper describes large-area molecular tunneling junctions comprising self-assembled monolayers (SAMs) of light-harvesting protein complexes using eutectic Ga-In (EGaIn) as a top contact. The complexes, which are readily isolable in large quantities from spinach leaves, self-assemble on top of SAMs of [6,6]-phenyl-C61-butyric acid (PCBA) on gold (Au) supported by mica substrates (AuMica), which induces them to adopt a preferred orientation with respect to the electron transport chain that runs across the short axis of each complex, leading to temperature-independent rectification. We compared trimeric protein complexes isolated from thermophilic cyanobacteria to monomeric complexes extracted from spinach leaves by measuring charge-transport at variable temperatures and over the course of at least three months. Transport is independent of temperature in the range of 130 to 310 K for both protein complexes, affirming that the likely mechanism is non-resonant tunneling. The junctions rectified current and were stable for at least three months when stored at room temperature in ambient conditions, with the yield of working junctions falling from 100% to 97% over that time. These results demonstrate a straightforward strategy for forming remarkably robust molecular junctions, avoiding the fragility that is common in molecular electronics.