Single-residue Resolution Research Articles

Protein Quakes in Redox Metalloenzymes: Clues to Molecular Enzyme Conductivity Triggered by Binding of Small Substrate Molecules.

Multicentre redox metalloproteins undergo conformational changes on electrochemical surfaces, or on enzyme substrate binding. The two-centre copper enzymes, laccase (Type I and TypeII/III Cu) and nitrite reductase (CuNIR) (Type I and Type II Cu) are examples. With some exceptions, these enzymes show no non-turnover voltammetry on Au(111)-surfaces modified by thiol based self-assembled molecular monolayers, but dioxygen or nitrite substrate triggers strong electrocatalytic signals. Scanning tunnelling microscopy also shows high conductivity only when dioxygen or nitrite is present. Atomic force microscopy shows constant CuNIR height but pronounced structural expansion in the electrocatalytic range on nitrite binding. We have recently offered a rationale, based on ab initio quantum chemical studies of water/nitrite substitution in a 740-atom CuNIR fragment. Presently we provide much more detailed structural assignment mapped to single-residue resolution. NO2 --binding induces both a 2 Å Cu-Cu distance increase, and pronounced frontier orbital delocalization strongly facilitating ET between the Cu regions. The conformational changes transmit from the catalytic Type II centre to the electron inlet Type I centre, via the His129-Cys130 ligands, and via Type I-Cys130 or Type I-His129 ending at Type II Asp92. The ET patterns are reflected in different atomic Mulliken charges in the water and nitrite CuNIR fragment.

The 2024 IPD-MHC database update: a comprehensive resource for major histocompatibility complex studies.

The IPD-MHC Database project (http://www.ebi.ac.uk/ipd/mhc/) serves as a comprehensive and expertly curated repository for major histocompatibility complex (MHC) sequences from non-human species, providing the necessary infrastructure and tools to study the function and evolution of this highly polymorphic genomic region. In its latest version, the IPD-MHC database has expanded both in content and in the tools for data visualization and comparison. The database now hosts over 18 000 MHC alleles from 125 species, organized into eleven taxonomic groups, all manually curated and named by the Comparative MHC Nomenclature Committee. A cetacean section has recently been included, offering researchers valuable data to study the immune system of whales, dolphins, and porpoises, as well establishing the official nomenclature platform for the Cetacea Leukocyte Antigens (CeLA). In response to user demand and reflecting broader trends in bioinformatics and immunogenetics, IPD-MHC now includes the predicted tertiary structure of over 8000 alleles and allows comparison and visualisation of allele variation within and between species at single residue resolution. These latest developments maintain the critically important link between official nomenclature of curated alleles and the ability to analyse this complex polymorphism using the most up to date methods within a single repository.

Widespread adaptive evolution in angiosperm photosystems provides insight into the evolution of photosystem II repair.

Oxygenic photosynthesis generates the initial energy source that fuels nearly all life on Earth. At the heart of the process are the photosystems, which are pigment binding multi-protein complexes that catalyse the first step of photochemical conversion of light energy into chemical energy. Here, we investigate the molecular evolution of the plastid-encoded photosystem subunits at single-residue resolution across 773 angiosperm species. We show that despite an extremely high level of conservation, 7% of residues in the photosystems, spanning all photosystem subunits, exhibit hallmarks of adaptive evolution. Through in silico modelling of these adaptive substitutions, we uncover the impact of these changes on the predicted properties of the photosystems, focussing on their effects on co-factor binding and inter-subunit interface formation. By analyzing these cohorts of changes, we reveal that evolution has repeatedly altered the interaction between photosystem II and its D1 subunit in a manner that is predicted to reduce the energetic barrier for D1 turnover and photosystem repair. Together, these results provide insight into the trajectory of photosystem adaptation during angiosperm evolution.

Structure-guided Evolutionary Analysis of Interactome Network Rewiring at Single Residue Resolution in Yeasts

Protein-protein interactions (PPIs) are known to rewire extensively during evolution leading to lineage-specific and species-specific changes in molecular processes. However, the detailed molecular evolutionary mechanisms underlying interactome network rewiring are not well-understood. Here, we combine high-confidence PPI data, high-resolution three-dimensional structures of protein complexes, and homology-based structural annotation transfer to construct structurally-resolved interactome networks for the two yeasts S. cerevisiae and S. pombe. We then classify PPIs according to whether they are preserved or different between the two yeast species and compare site-specific evolutionary rates of interfacial versus non-interfacial residues for these different categories of PPIs. We find that residues in PPI interfaces evolve significantly more slowly than non-interfacial residues when using lineage-specific measures of evolutionary rate, but not when using non-lineage-specific measures. Furthermore, both lineage-specific and non-lineage-specific evolutionary rate measures can distinguish interfacial residues from non-interfacial residues for preserved PPIs between the two yeasts, but only the lineage-specific measure is appropriate for rewired PPIs. Finally, both lineage-specific and non-lineage-specific evolutionary rate measures are appropriate for elucidating structural determinants of protein evolution for residues outside of PPI interfaces. Overall, our results demonstrate that unlike tertiary structures of single proteins, PPIs and PPI interfaces can be highly volatile in their evolution, thus requiring the use of lineage-specific measures when studying their evolution. These results yield insight into the evolutionary design principles of PPIs and the mechanisms by which interactions are preserved or rewired between species, improving our understanding of the molecular evolution of PPIs and PPI interfaces at the residue level.

Resolving the Amino Acid Sequence of Aβ1‐42 at the Single‐Residue Level Using Subnanopores in Ultrathin Films

AbstractProteoforms are proteins derived from highly related genes or post translational modifications (PTMs) of the same protein. They share extremely similar primary structures but have varying functions. Unfortunately, protein de novo sequencing including specific PTM/mutation detection is still challenging. Herein, a nanopore‐based technique is reported to resolve the amino acid order of amyloid‐β (Aβ1‐42) with site specificity. Subnanopores are sputtered in 5 nm‐thick inorganic membranes with a sensing depth of 0.66 nm inferred by finite element analysis. Denatured molecules at 0.45 ng mL−1 translocate through subnanopores while the current traces are sampled at 500 kHz with rms noise <15 pA. Hundreds of blockades are clustered using machine learning, and multiple blockades are averaged to establish current consensus. Consensus traces strongly correlate with a linear model of amino acid volume of Aβ1‐42 at single residue resolution, with Pearson Correlation Coefficients (PCCs) of 0.81 ± 0.03 and 0.92 ± 0.03 before and after dynamic time warping (DTW). A scrambled version of Aβ1‐42 is tested for validation purposes. Deep learning classification reveals that different polypeptides generate distinct translocation fluctuating patterns, but variations become imperceptible for the same species measured across nanopores (Area Under the Curve, AUC 0.93 ± 0.05 vs 0.64 ± 0.12). Lastly, important PTMs and mutations are site‐specifically located along the primary structure, implying new potential clinical applications.

Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3ζ protein variants

Tau protein is an intrinsically disordered protein that plays a key role in Alzheimer's disease (AD). In brains of AD patients, Tau occurs abnormally phosphorylated and aggregated in neurofibrillary tangles (NFTs). Together with Tau, 14-3-3 proteins – abundant cytosolic dimeric proteins – were found colocalized in the NFTs. However, so far, the molecular mechanism of the process leading to pathological changes in Tau structure as well as the direct involvement of 14-3-3 proteins are not well understood. Here, we aimed to reveal the effects of phosphorylation by protein kinase A (PKA) on Tau structural preferences and provide better insight into the interaction between Tau and 14-3-3 proteins. We also addressed the impact of monomerization-inducing phosphorylation of 14-3-3 at S58 on the binding to Tau protein. Using multidimensional nuclear magnetic resonance spectroscopy (NMR), chemical cross-linking analyzed by mass spectrometry (MS) and PAGE, we unveiled differences in their binding affinity, stoichiometry, and interfaces with single-residue resolution. We revealed that the interaction between 14-3-3 and Tau proteins is mediated not only via the 14-3-3 amphipathic binding grooves, but also via less specific interactions with 14-3-3 protein surface and, in the case of monomeric 14-3-3, also partially via the exposed dimeric interface. In addition, the hyperphosphorylation of Tau changes its affinity to 14-3-3 proteins. In conclusion, we propose quite complex interaction mode between the Tau and 14-3-3 proteins.

Quantitative characterization of peptide-lipid membrane interaction with single-residue resolution using coarse-grained MD simulations, AFM force spectroscopy, and theoretical modeling

Quantitative characterization of peptide-lipid membrane interaction with single-residue resolution using coarse-grained MD simulations, AFM force spectroscopy, and theoretical modeling

Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting.

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins

Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins

Saturation mutagenesis of α-synuclein reveals monomer fold that modulates aggregation.

α-Synuclein (aSyn) aggregation underlies neurodegenerative synucleinopathies. aSyn seeds are proposed to replicate and propagate neuronal pathology like prions. Seeding of aSyn can be recapitulated in cellular systems of aSyn aggregation; however, the mechanism of aSyn seeding and its regulation are not well understood. We developed an mEos-based aSyn seeding assay and performed saturation mutagenesis to identify with single-residue resolution positive and negative regulators of aSyn aggregation. We not only found the core regions that govern aSyn aggregation but also identified mutants outside of the core that enhance aggregation. We identified local structure within the N terminus of aSyn that hinders the fibrillization propensity of its aggregation-prone core. Based on the screen, we designed a minimal aSyn fragment that shows a ~4-fold enhancement in seeding activity and enabled discrimination of synucleinopathies. Our study expands the basic knowledge of aSyn aggregation and advances the design of cellular systems of aSyn aggregation to diagnose synucleinopathies based on protein conformation.

MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation

Knowing that an antibody's sensitivity and specificity is accurate is crucial for reliable data collection. This certainty is especially difficult to achieve for antibodies (Abs) which bind post-translationally modified proteins. Here we describe two validation methods using surrogate proteins in western blot and ELISA. The first method, which we termed “MILKSHAKE” is a modified maltose binding protein, hence the name, that is enzymatically conjugated to a peptide from the chosen target which is either modified or non-modified at the residue of interest. The surety of the residue's modification status can be used to confirm Ab specificity to the target's post-translational modification (PTM). The second method uses a set of surrogate proteins, which we termed “Sundae”. Sundae consists of a set of modified maltose binding proteins with a genetically encoded target sequence, each of which contains a single amino acid substitution at one position of interest. With Sundae, Abs can be evaluated for binding specificities to all twenty amino acids at a single position. Combining MILKSHAKE and Sundae methods, Ab specificity can be determined at a single-residue resolution. These data improve evaluation of commercially available Abs and identify off-target effects for Research-Use-Only and therapeutic Abs.

Deafness-Associated ADGRV1 Mutation Impairs USH2A Stability through Improper Phosphorylation of WHRN and WDSUB1 Recruitment.

The ankle-link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness-associated human Y6244fsX1 mutation, the authors show herethat the Y6236fsX1 mutation disrupts the interaction between adhesion G protein-coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP-PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two-hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation-dependent manner. Further FlAsH-BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single-residue resolution. In conclusion, the present data suggest that ALC organizationand accompanying local signal transduction play important roles in regulating the stability of the ALC.

Specific phosphorylation of microtubule-associated protein 2c by extracellular signal–regulated kinase reduces interactions at its Pro-rich regions

Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal–regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.

Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation

BackgroundCurrent understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans.ResultsWe show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86–93: P83QQPPPP93, and 166–175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200–250 adopt three partially populated α-helices. Residues 345–355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids.ConclusionOur NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein–protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones.AreasBiophysics, Structural Biology, Biochemistry & Neurosciences.

Ribosome exit tunnel electrostatics.

The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in rabbit reticulocytes. Here we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show that a strong electrostatic screening is due to water molecules (not mobile ions) attracted to the ribosomal nucleic acid phosphate moieties buried in the immediate vicinity of the tunnel wall. We also show how the tunnel wall components and local ribosomal protein protrusions impact on the electrostatic potential profile and impede charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of -40% to +85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derive, at single-residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall, shape, and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, x-ray crystallography, physical chemistry, biomechanics, and synthetic and multiomics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control, and the role of the ribosome's mechanochemistry in the cotranslational protein folding.

Amber Suppression Technology for Mapping Site-specific Viral-host Protein Interactions in Mammalian Cells.

Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinity-purified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions. Graphic abstract: Schematic overview of the virus-host interaction assay based on an amber suppression approach. Mammalian cells grown in Bpa-supplemented medium are co-transfected with plasmids encoding viral sequences carrying a Flag tag, a (TAG) stop codon at the desired position, and an amber suppressor tRNA (tRNACUA)/aminoacyl tRNA synthetase (aaRS) orthogonal pair. Cells are then exposed to UV, to generate protein-protein crosslinks, followed by immunoprecipitation with anti-Flag magnetic beads. The affinity-purified crosslinks are probed by western blot using an anti-Flag antibody and the crosslinked host proteins are characterised by mass spectrometry.

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) – haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb – Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

Single Nucleotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.

RNA–protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA–protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g., in cell or tissue samples. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide–RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18O4-γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γ-phosphate to the 5′-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.

Multiple-State Monitoring of SOD1 Amyloid Formation at Single-Residue Resolution by Rheo-NMR Spectroscopy.

Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1-infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.