The binding specificity of OppA determines the selectivity of the oligopeptide ATP-binding cassette transporter

The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.     

On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis

Lactococcus lactis degrades exogenous proteins such as beta-casein to peptides of 4-30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppA(Ll)) of the oligopeptide transport system (Opp) of L.LACTIS: has the unique capacity to bind peptides from five up to at least 20 residues. To study the binding mechanism of OppA(Ll), nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non-fluorescent or bulky and fluorescent groups. Also, nonameric peptides with a non-natural residue, azatryptophan, at positions 3 or 7 were used. The fluorescence of azatryptophan reports on the polarity of the environment. The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein. The peptide binding mechanism of OppA(Ll) is discussed in relation to known three-dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.     

Escherichia coli peptide binding protein OppA has a preference for positively charged peptides

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.     

Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis

The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar K(m) for transport, whereas the V(max) was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.     

Specificity and interactions of the protein OppA: partitioning solvent binding effects using mass spectrometry

Mass spectrometry (MS) was used to characterise the binding of the 58 kDa protein OppA to 11 peptides with diverse properties. Peptides with two, three and five amino acid residues were added to OppA, and the mass spectra showed that the highest-affinity complexes are formed between OppA and tripeptide ligands. Lower-affinity complexes were observed for OppA and dipeptide ligands, and no complex formation was detected with pentapeptides or a tripeptide in which the N-terminal amino group was acetylated. Tripeptides containing a single d amino acid residue were found not to bind to native OppA. Evidence from the peak width and the, charge in the spectra of the complexes suggests that the bound peptides are encapsulated by the protein in a solvent-filled cavity in the gas phase of the mass spectrometer. Analysis of the proportions of peptide-bound and free proteins under low-energy MS conditions shows a good correlation with solution-phase K(d) measurements where available. Increasing the internal energy of the gas-phase complex led to dissociation of the complex. The ease of dissociation is interpreted in terms of the intrinsic stability of the complex in the absence of the stabilising effects of bulk solvent. The results from this study demonstrate insensitivity to the hydrophobic and ionic properties, of the side-chains of the peptides, in contrast to the investigation of other protein ligand systems by MS. Moreover, these findings are in accord with the physiological role of this protein in allowing into the cell di- and tripeptides containing naturally occurring amino acids, regardless of their sequence, while barring access to potentially harmful peptide mimics.     

Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.     

Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils

Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This further validates the hypothesis that the favorable tertiary interactions in protein systems such as merR go a long way in stabilizing nonnatural coordination environments in biological systems. Similarly, for the binding of Cd(II) to the TRI family, the dissociation constants range from 1.3 x 10(-)(6) M for Baby L9C to 8.3 x 10(-)(9) M for TRI L9C, showing a similar nature of stable aggregate formation.     

Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.     

Thermodynamics of peptide binding to the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP plays an essential role in antigen processing and immune response to infected or malignant cells. TAP translocates proteasomal degradation products from the cytosol into the endoplasmic reticulum, where MHC class I molecules are loaded with these peptides. Kinetically stable peptide-MHC complexes are transported to the cell surface for inspection by cytotoxic T lymphocytes. The transport cycle of TAP is initiated by peptide binding, which is responsible for peptide selection and for stimulation of ATP-hydrolysis and subsequent translocation. Here we have analysed the driving forces for the formation of the peptide-TAP complex by kinetic and thermodynamic methods. First, the apparent peptide association and dissociation rates were determined at various temperatures. Strikingly, very high activation energies for apparent association (E(a)(ass)=106 kJmol(-1)) and dissociation (E(a)(diss)=80 kJmol(-1)) of the peptide-TAP complex were found. Next, the temperature-dependence of the peptide affinity constants was investigated by equilibrium-binding assays. Along with calculations of free enthalpy deltaG, enthalpy deltaH and entropy deltaS, a large positive change in heat capacity was resolved (deltaC degrees =23 kJmol(-1)K(-1)), indicating a fundamental structural reorganization of the TAP complex upon peptide binding. The inspection of the conformational entropy reveals that approximately one-fourth of all TAP residues is rearranged. These thermodynamic studies indicate that at physiological temperature, peptide binding is endothermic and driven by entropy.     

Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.     

Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein

The Nef proteins of simian and human immunodeficiency viruses are known to directly bind and downregulate the CD4 receptor of infected cells. Recent results suggest that residues forming an alpha-helix N-cap in the CD4 cytoplasmic domain play a role in binding of CD4 to human immunodeficiency virus type 1 Nef protein. We determined the dissociation constants between Nef and several CD4 peptides that contain or do not contain the respective alpha-helix N-cap. Further, we compared helical secondary structure content of these CD4 peptide variants by circular dichroism spectroscopy. We conclude that presence of an alpha-helix in CD4 cytoplasmic domain increases CD4 affinity to Nef. In addition, the amino acid sequence of residues forming the helix N-cap influences CD4 affinity to Nef, too. Finally, the structural changes induced in Nef and CD4 upon binding to each other are investigated.     

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.     

Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions

The fine modulation of peptide–antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125–136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore^TM, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope results in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide–antibody interactions.     

Crystallographic and calorimetric analysis of peptide binding to OppA protein.

Isothermal titration calorimetry has been used to study the binding of 20 different peptides to the peptide binding protein OppA, and the crystal structures of the ligand complexes have been refined. This periplasmic binding protein, part of the oligopeptide permease system of Gram negative bacteria, has evolved to bind and enclose small peptides of widely varying sequences. The peptides used in this study have the sequence Lys-X-Lys, where X is any of the 20 commonly occurring amino acids. The various side-chains found at position 2 on the ligand fit into a hydrated pocket. The majority of side-chains are restrained to particular conformations within the pocket. Water molecules act as flexible adapters, matching the hydrogen-bonding requirements of the protein and ligand and shielding charges on the buried ligand. This use of water by OppA to broaden the repertoire of its binding site is not unique, but contrasts sharply with other proteins which use water to help bind ligands highly selectively. Predicting the thermodynamics of binding from the structure of the complexes is highly complicated by the influence of water on the system.     

Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance.

Differences in the affinity of a monoclonal antibody raised against the protein of tobacco mosaic virus for 15 related peptides (residues 134-146) carrying single-residue modifications were investigated using a novel biosensor technology (Pharmacia BIAcore). Analysis of the peptide-antibody interaction in real time allowed fast and reproducible measurements of both association and dissociation rate constants. Out of 15 mutant peptides analyzed, five were not recognized by the antibody at all, and seven were recognized as well as the wild-type peptide. For three of the peptides, the rate constants were different for the mutant and wild-type peptides. The pattern of residue recognition suggests that the epitope is formed by three residues (140, 143, and 144) in a helical conformation that mimics the structure in the protein. Even a minor modification of these residues totally abolishes recognition by the antibody. Modifications of adjacent residues result in small but significant differences in association and/or dissociation rate constants. One of the recognized residues is totally buried in the three-dimensional structure of TMV protein, suggesting that a structural rearrangement next to the helix occurs during protein-antibody interaction.     

Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule

Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.     

The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.     

Protein structural requirements and properties of membrane binding by gamma-carboxyglutamic acid-containing plasma proteins and peptides

The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.     

Binding of hormones and neuropeptides by calmodulin

Calmodulin exhibits high-affinity, calcium-dependent binding of 1 mol/mol of the vasoactive intestinal peptide (VIP), secretin, and either the 42- or 43-residue gastric inhibitory peptide (GIP) with dissociation constants of 0.05-0.14 microM. The affinity of VIP for calmodulin approaches its affinity for the cell-surface VIP receptors. These peptides compete with both smooth muscle myosin light chain kinase and glucagon in calmodulin binding. Calculation of amino acid frequencies for eight calmodulin binding peptides (VIP, GIP, secretin, ACTH, beta-endorphin, substance P, glucagon, and dynorphin [Malencik, D. A., & Anderson, S. R. (1982) Biochemistry 21, 3480]) shows a below-average incidence of glutamyl residues, above-average incidence of glutaminyl residues, and average incidence of both aspartyl and asparaginyl residues. Predictions of structure from sequence suggest that the bound peptides contain strongly basic turns and coils in close association with regions having above-average beta-sheet potential. The temperature dependence of glucagon binding by calmodulin shows that the association is enthalpy driven.     

High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR

Immunogenic peptides have been shown to bind detergent-solubilized class II (Ia) molecules from mice. In this investigation, we report that highly purified HLA-DR (DR) molecules in detergent solution are capable of binding a synthetic peptide (HAp) derived from the influenza hemagglutinin sequence. Although the presentation of this peptide has been demonstrated only to DR1-restricted Th cells, the association rate constants for the formation of HAp-DR1, -DR5, and -DR8 complexes were essentially identical (ka = 1.1 x 10(2) to 1.6 x 10(2) M-1 s-1). By contrast, the value of the rate constants for the dissociation of preformed HAp-DR1, -DR5, and -DR8 complexes varied nearly threefold (kd = 1.6 x 10(6) to 4.4 x 10(-6) s-1). The value of the equilibrium dissociation constants (KD) derived from these rate constants were 13 nM, 24 nM, and 28 nM, for HAp-DR1, -DR5, and -DR8 complexes, respectively. Scatchard analysis demonstrated that the KD obtained from the rate constants for the HAp-DR1 reaction was in excellent agreement with that obtained under equilibrium conditions. SDS-PAGE confirmed that the HAp-DR complexes were remarkably stable, as HAp remained associated with the DR alpha beta heterodimer after treatment of the complexes with SDS and beta-mercaptoethanol. Steady-state binding studies demonstrated that 18% of all DR1 molecules had bound HAp at equilibrium, whereas only 3.8% of all DR8 molecules had bound HAp under identical conditions. The slight differences in the KD for HAp-DR complexes suggest that differences in the affinity of a peptide for DR alleles alone may not always explain the process of MHC restriction.