Evidence for conformationally different states of interleukin‐10: binding of a neutralizing antibody enhances accessibility of a hidden epitope

We present the mapping of two anti‐human interleukin‐10 (hIL‐10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL‐10‐derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L ‐amino acids. The epitope‐derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody–hIL‐10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X‐ray structure of IL‐10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL‐10 can adopt a conformational state other than that observed in the crystal structure. Copyright © 1999 John Wiley & Sons, Ltd.     

Structural and molecular analysis of a protective epitope of Lyme disease antigen OspA and antibody interactions

The murine monoclonal antibody LA‐2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi , the causative agent of Lyme disease in North America. Human antibody equivalence to LA‐2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA‐2 protective epitope is important for developing OspA‐based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure‐based analysis of the LA‐2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA‐2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA‐2 light chain, N52 on the LA‐2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA‐2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti‐tau antibody AT8

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Detection of oxytocin,atrial natriuretic peptide,and brain natriuretic peptide using novel imprinted polymers produced with amphiphilic monomers

The cystine‐bridged cyclic peptide hormones (CBCPHs) represent signature structural feature as well as unique biological activity. In this study, three CBCPHs have been identified and characterized, namely, oxytocin, atrial natriuretic peptides (ANPs), and brain natriuretic peptides (BNPs). Because research has shown that ANPs and BNPs are powerful diagnostic biomarkers for heart disease, a highly laudable endeavor would be to develop a novel sensor for detecting ANP or BNP levels. Therefore, an amphiphilic monomer Acr‐His‐NHNH‐Fmoc was synthesized to form molecularly imprinted polymers (MIPs) for targeted CBCPH detection. First, oxytocin, a cardiovascular hormone and a CBCPH, was used as a template to fabricate MIPs on quartz crystal microbalance (QCM) chips. On the other hand, fabricated selected ANP segment or BNP segment as an epitope is able to construct epitope‐mediated MIPs (EMIPs) for ANP or BNP. The developed oxytocin or ANP sensor reached a detection limitation of 0.1nM with the dissociation constants being 30pM for oxytocin and 20pM for ANP. Moreover, BNP sensor achieved a detection limitation of 2.89pM with an even lower K_d value as 2pM. Compared with the performance of EMIPs, the imprinted films showed high affinity and selectivity in special binding to CBCPHs. The developed MIPs‐QCM biosensors thus provide an improved sensing platform using an amphiphilic monomer and may be useful for applications toward cyclotides, cystine knot motifs, or insulin‐like peptides.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

Epitope characterization of an anti‐PD‐L1 antibody using orthogonal approaches

The binding of programmed death ligand 1 protein (PD‐L1) to its receptor programmed death protein 1 (PD‐1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD‐L1/PD‐1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD‐L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD‐L1. Hydrogen–deuterium exchange mass spectrometry (HDX‐MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD‐1 binding interface, consistent with a desired PD‐1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD‐L1 variants confirmed that the region identified by HDX‐MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD‐L1‐mediated immune evasion. This is the first report of an epitope for any antibody targeting PD‐L1 and demonstrates the power of combining orthogonal epitope mapping techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of a diagnostic Fab fragment binding trimeric Lewis X

Lewis X trisaccharides normally function as essential cell–cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54‐5C10‐A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 Å crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291‐2G3‐A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54‐5C10‐A. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

B‐type natriuretic peptide and extracellular matrix protein interactions in human cardiac fibroblasts

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B‐type natriuretic peptide (BNP) is anti‐fibrotic, inhibits collagen production, augments matrix metalloproteinases, and suppresses CF proliferation. Recently, we demonstrated that the ECM protein fibronectin (FN) augmented production of BNP's second messenger, 3′, 5′ cyclic guanosine monophosphate (cGMP) in CFs, supporting crosstalk between FN, BNP, and its receptor, natriuretic peptide receptor A (NPR‐A). Here, we address the specificity of FN to augment cGMP generation by investigating other matrix proteins, including collagen IV which contains RGD motifs and collagen I and poly‐L ‐lysine, which have no RGD domain. Collagen IV showed increased cGMP generation to BNP similar to FN. Collagen I and poly‐L ‐lysine had no effect. As FN also interacts with integrins, we then examined the effect of integrin receptor antibody blockade on BNP‐mediated cGMP production. On FN plates, antibodies blocking RGD‐binding domains of several integrin subtypes had little effect, while a non‐RGD domain interfering integrin αvβ3 antibody augmented cGMP production. Further, on uncoated plates, integrin αvβ3 blockade continued to potentiate the BNP/cGMP response. These studies suggest that both RGD containing ECM proteins and integrins may interact with BNP/NPR‐A to modulate cGMP generation. J. Cell. Physiol. 225: 251–255, 2010. © 2010 Wiley‐Liss, Inc.     

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures

The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity‐adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody–epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)‐containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody–epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His‐tag‐containing recombinant human glucose‐6‐phosphate isomerase in combination with an anti‐His‐tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody–epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto‐antigen in combination with a rabbit polyclonal anti‐TRIM21 antibody. Peptide chip analysis showed that antibody–epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA‐containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody–antigen complexes under neutral pH and low salt conditions for subsequent investigations. Copyright © 2014 John Wiley & Sons, Ltd.     

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope‐scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope‐scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody‐binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope‐scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV‐1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope‐scaffold that bound 2F5 with subnanomolar affinity (K_D = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope‐scaffold represents a successful example of rational protein backbone engineering and protein–protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. Proteins 2014; 82:2770–2782. © 2014 Wiley Periodicals, Inc.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non‐human primate models

Antibody based manipulation of the CD137 (4‐1BB) co‐signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti‐human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti‐CD137 mAb (4B4‐1), recognized activated PBMCs from both human and non‐human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species (～95% identical), with the extracellular domain differing by only 9–10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co‐stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody‐binding protein as an adapter can simplify antibody functionalization by forming a specific antibody‐bound complex and introducing site‐specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody‐binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein (“lasso”), which binds human immunoglobulin G1 (IgG1) with K _D = 0.53 n m and a dissociation rate that is 55‐ to 84‐fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme‐linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12‐fold. The small size of the lasso and a long half‐life of dissociation make the peptide a useful tool in antibody detection and immobilization.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Structural studies of the tethered N‐terminus of the Alzheimer's disease amyloid‐β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ_1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.     

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

Humanin (HN) is a linear 24‐aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß‐amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1–40) and characterization of the interaction structure through a molecular modeling study. Wild‐type HN and HN‐sequence mutations were synthesized by SPPS and the HPLC‐purified peptides characterized by MALDI‐MS. The interaction epitopes between HN and Aß(1–40) were identified by affinity‐MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity‐bound peptides. The affinity‐MS analyses revealed HN(5–15) as the epitope sequence of HN, whereas Aß(17–28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1–40) and by ELISA with Aß(1–40) and Aß‐partial sequences as ligands to immobilized HN. The specificity and affinity of the HN‐Aß interaction were characterized by direct ESI‐MS of the HN‐Aß(1–40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K_D of the complex of 610 n m . A molecular dynamics simulation of the HN‐Aß(1–40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1–40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.