Interaction of epitope-related and -unrelated peptides with anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment

The binding of four epitope-related peptides and three library-derived, epitope-unrelated peptides of different lengths (10-14 amino acids) and sequence by anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment was studied by isothermal titration calorimetry. The binding constants K(A) at 25 degrees C vary between 5.1 x 10(7) M (-1) for the strongest and 1.4 x 10(5) M (-1) for the weakest binder. For each of the peptides complex formation is enthalpically driven and connected with unfavorable entropic contributions; however, the ratio of enthalpy and entropy contributions to deltaG(0) differs markedly for the individual peptides. A plot of -deltaH(0) vs -TdeltaS(0) shows a linear correlation of the data for a wide variety of experimental conditions as expected for a process with deltaC(p) much larger than deltaS(0). The dissimilarity of deltaC(p) and deltaS(0) also explains why deltaH(0) and TdeltaS(0) show similar temperature dependences resulting in relatively small changes of deltaG(0) with temperature. The heat capacity changes deltaC(p) upon antibody-peptide complex formation determined for three selected peptides vary only in a small range, indicating basic thermodynamic similarity despite different key residues interacting in the complexes. Furthermore, the comparison of van't Hoff and calorimetric enthalpies point to a non-two-state binding mechanism. Protonation effects were excluded by measurements in buffers of different ionization enthalpies. Differences in the solution conformation of the peptides as demonstrated by circular dichroic measurements do not explain different binding affinities of the peptides; specifically a high helix content in solution is not essential for high binding affinity despite the helical epitope conformation in the crystal structure of p24.     

Interaction of FliS flagellar chaperone with flagellin

Premature polymerization of flagellin (FliC), the main component of flagellar filaments, is prevented by the FliS chaperone in the cytosol. Interaction of FliS with flagellin was characterized by isothermal titration calorimetry producing an association constant of 1.9x10(7) M-1 and a binding stoichiometry of 1:1. Experiments with truncated FliC fragments demonstrated that the C-terminal disordered region of flagellin is essential for FliS binding. As revealed by thermal unfolding experiments, FliS does not function as an antifolding factor keeping flagellin in a secretion-competent conformation. Instead, FliS binding facilitates the formation of alpha-helical secondary structure in the chaperone binding region of flagellin.     

A simple novel method for determination of an inhibition constant by isothermal titration microcalorimetry. The effect of fluoride ion on urease

Abstract

A simple novel method was introduced for determination of an inhibitor binding constant (Kj) and enthalpy of binding by isothermal titration microcalorimetry technique. This method was applied to the binding of fluoride ion, as an inhibitor, with the active sites of jack bean urease at pH = 7.0 (Tris 30 mM) and T = 300°K. The dissociation equilibrium constant measured by this method was markedly consistent with the inhibition constant obtained from assay of enzyme activity in the presence of fluoride ion.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291–L301, has an average helicity greater than 60% and the C-terminal segment, from S304–L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (> 90%), even at non-interacting sites, for c-Myb homologues.     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding

The oligomerization and aggregation of the amyloid-β (Aβ) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of Aβ-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying Aβ interactions is currently very limited. We have earlier determined the structure of Aβ(1-40) in complex with the affibody protein Z_Aβ3, a selected binding protein based on a three-helix bundle scaffold (Z domain). Z_Aβ3 is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z_Aβ3 binds to the central and C-terminal part of Aβ (residues 17-36), which adopts a β-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z_Aβ3:Aβ(1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z_Aβ3 is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z_Aβ3:Aβ(1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both Aβ and Z_Aβ3 fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and Aβ is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of Aβ. Options for improvement of Z_Aβ binding proteins are discussed.     

Structural Mimicry of O-Antigen by a Peptide Revealed in a Complex with an Antibody Raised against Shigella flexneri Serotype 2a

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.     

Bile acid interactions with rabbit ileal lipid binding protein and an engineered helixless variant reveal novel ligand binding properties of a versatile beta-clam shell protein scaffold

The intracellular ileal lipid binding proteins (ILBPs) are involved in the transport and enterohepatic circulation of bile acids. ILBPs from different species show high sequence and structural homology and have been shown to bind multiple bile acid ligands with differing degrees of selectivity and positive co-operativity. Human ILBP binds bile acid derivatives in a well-characterised 2:1 ligand:protein complex, however, we show that the highly homologous rabbit ILBP (82% sequence identity) with seven conservative substitutions preferentially binds multiple conjugated deoxycholate ligands in a novel 3:1 binding mode essentially within the same beta-clam shell structure. We have extended these studies to investigate the role of the alpha-helical capping motif (residues 9-35) in controlling the dimensions of the binding cavity and ligand uptake. Substituting the alpha-helical motif (residues 9-35) with a short Gly-Gly-Ser-Gly linker dramatically affects the protein stability such that under physiological conditions the mutant (Deltaalpha-ILBP) is highly disordered. However, we show that the inability of the mutant to adopt a stable three-dimensional structure under these conditions is no barrier to binding ligands with near-native affinity. These structural modifications not only demonstrate the possibility of strong coupling between ligand binding and protein folding, but result in changes in bile acid selectivity and binding stoichiometry, which we characterise in detail using isothermal calorimetry and mass spectrometry.     

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the α-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the “unbound” and “bound” states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2∼P7∼P10 > P9∼P6 > P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.     

Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr^9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPr^sc. By using fluorescence and circular dichroism, we first determined qualitatively that HPr^sc and HPr^9-30 did bind to EI^sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr^9-30 and HPr^sc for EI^sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr^9-30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI^sc and HPr^sc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr^sc for EI^sc was smaller than in other species. However, the affinity of HPr^9-30 for EI^sc was only moderately lower than that of EI^sc for HPr^sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.     

Structural insights into recognition of acetylated histone ligands by the BRPF1 bromodomain

Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ HAT complex and contains a unique combination of domains typically found in chromatin-associated factors, which include plant homeodomain (PHD) fingers, a bromodomain and a proline-tryptophan-tryptophan-proline (PWWP) domain. Bromodomains are conserved structural motifs generally known to recognize acetylated histones, and the BRPF1 bromodomain preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We solved the X-ray crystal structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptides. Site-directed mutagenesis on residues in the BRPF1 bromodomain-binding pocket was carried out to investigate the contribution of specific amino acids on ligand binding. Our results provide critical insights into the molecular mechanism of ligand binding by the BRPF1 bromodomain, and reveal that ordered water molecules are an essential component driving ligand recognition.     

Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%–36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%–50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a K_a of 5.39 ± 0.45 × 10³–7.56 ± 1.47 × 10³ M^− 1, and the catalytic domain of SlMan bound bivalent ions with a K_a of 1.06 ± 0.34 × 10³–3.86 ± 0.94 × 10³ M^− 1. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.