Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

The Activation Mechanism of the Insulin Receptor: A Structural Perspective.

The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.

A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase.

Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody.

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.

A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication.

Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.

Ice-Binding Proteins and Their Function.

Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.

A tamoxifen receptor within a voltage-gated sodium channel.

Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

A tri-functional amino acid enables mapping of binding sites for posttranslational-modification-mediated protein-protein interactions.

Posttranslational modification (PTM), through the recruitment of effector proteins (i.e., "readers") that signal downstream events, plays key roles in regulating a variety of cellular processes. To understand how a PTM is recognized, it is necessary to find its readers and, importantly, the location of the binding pockets responsible for PTM recognition. Although various methods have been developed to identify PTM readers, it remains a challenge to directly map the PTM-binding regions, especially for intrinsically disordered domains. Here, we demonstrate a photo-crosslinkable, clickable, and cleavable tri-functional amino acid, ADdis-Cys, that when coupled with mass spectrometry (ADdis-Cys-MS) can not only identify PTM readers from complex proteomes but also simultaneously map their PTM-recognition modules. Using ADdis-Cys-MS, we successfully identify the binding sites of several reader-PTM interactions, among which we discover human C1QBP as a histone chaperone. This robust method should find wide applications in examining other histone or non-histone PTM-mediated protein-protein interactions.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

The CH1 domain influences the expression and antigen sensing of the HIV-specific CH31 IgM-BCR and IgG-BCR.

How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here, we study antigen binding and sensing of human Ramos B cells expressing a BCR of either the IgM or IgG1 class with specificity for the CD4-binding-site of the envelope (Env) protein of the HIV-1. Both BCRs carry an identical antigen binding site derived from the broad neutralizing antibody (bnAb) CH31. We find a five times higher expression of the IgG1-BCR in comparison to the IgM-BCR on the surface of transfected Ramos B cells. The two BCR classes also differ from each other in their interaction with cognate HIV Env antigens in that the IgG1-BCR and IgM-BCR bind preferentially to polyvalent and monovalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR, we found that the class-specific BCR expression and antigen-sensing behavior can be transferred with the CH1γ domain from the IgG1-BCR to the IgM-BCR. Thus, the class of CH1 domain has an impact on BCR assembly and expression as well as on antigen sensing.

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

EGPDI: identifying protein-DNA binding sites based on multi-view graph embedding fusion.

Mechanisms of protein-DNA interactions are involved in a wide range of biological activities and processes. Accurately identifying binding sites between proteins and DNA is crucial for analyzing genetic material, exploring protein functions, and designing novel drugs. In recent years, several computational methods have been proposed as alternatives to time-consuming and expensive traditional experiments. However, accurately predicting protein-DNA binding sites still remains a challenge. Existing computational methods often rely on handcrafted features and a single-model architecture, leaving room for improvement. We propose a novel computational method, called EGPDI, based on multi-view graph embedding fusion. This approach involves the integration of Equivariant Graph Neural Networks (EGNN) and Graph Convolutional Networks II (GCNII), independently configured to profoundly mine the global and local node embedding representations. An advanced gated multi-head attention mechanism is subsequently employed to capture the attention weights of the dual embedding representations, thereby facilitating the integration of node features. Besides, extra node features from protein language models are introduced to provide more structural information. To our knowledge, this is the first time that multi-view graph embedding fusion has been applied to the task of protein-DNA binding site prediction. The results of five-fold cross-validation and independent testing demonstrate that EGPDI outperforms state-of-the-art methods. Further comparative experiments and case studies also verify the superiority and generalization ability of EGPDI.

Dynamic extracellular vestibule of human SERT: Unveiling druggable potential with high-affinity allosteric inhibitors.

The serotonin transporter (SERT) tightly regulates synaptic serotonin levels and has been the primary target of antidepressants. Binding of inhibitors to the allosteric site of human SERT (hSERT) impedes the dissociation of antidepressants bound at the central site and may enhance the efficacy of such antidepressants to potentially reduce their dosage and side effects. Here, we report the identification of a series of high-affinity allosteric inhibitors of hSERT in a unique scaffold, with the lead compound, Lu AF88273 (3-(1-(2-(1H-indol-3-yl)ethyl)piperidin-4-yl)-6-chloro-1H-indole), having 2.1 nM allosteric potency in inhibiting imipramine dissociation. In addition, we find that Lu AF88273 also inhibits serotonin transport in a noncompetitive manner. The binding pose of Lu AF88273 in the allosteric site of hSERT is determined with extensive molecular dynamics simulations and rigorous absolute binding free energy perturbation (FEP) calculations, which show that a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose are validated by site-directed mutagenesis and can explain much of the structure-activity relationship of these inhibitors using the relative binding FEP calculations. Together, our findings provide a promising lead compound and the structural basis for the development of allosteric drugs targeting hSERT. Further, they demonstrate that the divergent allosteric sites of neurotransmitter transporters can be selectively targeted.

Genome-wide maps of rare and atypical UV photoproducts reveal distinct patterns of damage formation and mutagenesis in yeast chromatin.

Ultraviolet (UV) light induces different classes of mutagenic photoproducts in DNA, namely cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and atypical thymine-adenine photoproducts (TA-PPs). CPD formation is modulated by nucleosomes and transcription factors (TFs), which has important ramifications for Ultraviolet (UV) mutagenesis. How chromatin affects the formation of 6-4PPs and TA-PPs is unclear. Here, we use UV damage endonuclease-sequencing (UVDE-seq) to map these UV photoproducts across the yeast genome. Our results indicate that nucleosomes, the fundamental building block of chromatin, have opposing effects on photoproduct formation. Nucleosomes induce CPDs and 6-4PPs at outward rotational settings in nucleosomal DNA but suppress TA-PPs at these settings. Our data also indicate that DNA binding by different classes of yeast TFs causes lesion-specific hotspots of 6-4PPs or TA-PPs. For example, DNA binding by the TF Rap1 generally suppresses CPD and 6-4PP formation but induces a TA-PP hotspot. Finally, we show that 6-4PP formation is strongly induced at the binding sites of TATA-binding protein (TBP), which is correlated with higher mutation rates in UV-exposed yeast. These results indicate that the formation of 6-4PPs and TA-PPs is modulated by chromatin differently than CPDs and that this may have important implications for UV mutagenesis.

Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome.

The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs.

Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal.

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. Reporter constructs, cell culture assays and computational modeling have made major contributions to answering this question, but analysis of elements in their natural context is an important complement. Here, we mutate Notch-dependent LAG-1 binding sites (LBSs) in the endogenous Caenorhabditis elegans sygl-1 gene, which encodes a key stem cell regulator, and analyze the consequences on sygl-1 expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of one LBS in a three-element cluster approximately halved both expression and stem cell pool size, whereas mutation of two LBSs essentially abolished them. Heterozygous LBS mutant clusters provided intermediate values. Our results lead to two major conclusions. First, both LBS number and configuration impact cluster activity: LBSs act additively in trans and synergistically in cis. Second, the SYGL-1 gradient promotes self-renewal above its functional threshold and triggers differentiation below the threshold. Our approach of coupling CRISPR/Cas9 LBS mutations with effects on both molecular and biological readouts establishes a powerful model for in vivo analyses of DNA cis-regulatory elements.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in the presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc. IMPORTANCE: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.

Identification of metal ion-binding sites in RNA structures using deep learning method.

Metal ion is an indispensable factor for the proper folding, structural stability and functioning of RNA molecules. However, it is very difficult for experimental methods to detect them in RNAs. With the increase of experimentally resolved RNA structures, it becomes possible to identify the metal ion-binding sites in RNA structures through in-silico methods. Here, we propose an approach called Metal3DRNA to identify the binding sites of the most common metal ions (Mg2+, Na+ and K+) in RNA structures by using a three-dimensional convolutional neural network model. The negative samples, screened out based on the analysis for binding surroundings of metal ions, are more like positive ones than the randomly selected ones, which are beneficial to a powerful predictor construction. The microenvironments of the spatial distributions of C, O, N and P atoms around a sample are extracted as features. Metal3DRNA shows a promising prediction power, generally surpassing the state-of-the-art methods FEATURE and MetalionRNA. Finally, utilizing the visualization method, we inspect the contributions of nucleotide atoms to the classification in several cases, which provides a visualization that helps to comprehend the model. The method will be helpful for RNA structure prediction and dynamics simulation study. Availability and implementation: The source code is available at https://github.com/ChunhuaLiLab/Metal3DRNA.

Quantitative model for genome-wide cyclic AMP receptor protein binding site identification and characteristic analysis.

Cyclic AMP receptor proteins (CRPs) are important transcription regulators in many species. The prediction of CRP-binding sites was mainly based on position-weighted matrixes (PWMs). Traditional prediction methods only considered known binding motifs, and their ability to discover inflexible binding patterns was limited. Thus, a novel CRP-binding site prediction model called CRPBSFinder was developed in this research, which combined the hidden Markov model, knowledge-based PWMs and structure-based binding affinity matrixes. We trained this model using validated CRP-binding data from Escherichia coli and evaluated it with computational and experimental methods. The result shows that the model not only can provide higher prediction performance than a classic method but also quantitatively indicates the binding affinity of transcription factor binding sites by prediction scores. The prediction result included not only the most knowns regulated genes but also 1089 novel CRP-regulated genes. The major regulatory roles of CRPs were divided into four classes: carbohydrate metabolism, organic acid metabolism, nitrogen compound metabolism and cellular transport. Several novel functions were also discovered, including heterocycle metabolic and response to stimulus. Based on the functional similarity of homologous CRPs, we applied the model to 35 other species. The prediction tool and the prediction results are online and are available at: https://awi.cuhk.edu.cn/∼CRPBSFinder.