影响多肽与花粉钙调素亲和性的因素──多肽的圆二色性及核磁共振研究

钙调素 ( Ca M)存在于所有真核细胞生物体内 ,它可与很多天然的生物活性肽结合 ,如 β-内啡肽、胰高血糖素和某些昆虫毒液肽等 [1] .有关 Ca M与多肽相互作用的研究普遍认为 ,对 Ca M有高亲和性的多肽应该具有形成α螺旋结构的显著倾向[2 ] .为进一步确认多肽的主链构象对 Ca M亲和能力的影响 ,我们采用圆二色性光谱和核磁共振波谱分析了荞麦花粉碱性十二肽 BPP-1和它的类似物 BPP-3的结构特征 ,配合多肽对花粉钙调素 ( p Ca M)的结合能力 ,发现肽链的可塑性和 C端的极性是影响多肽与 p Ca M亲和能力的因素 ,而形成 α螺旋结构的倾…     

Structure-based design and confirmation of peptide ligands for neuronal polo-like kinase to promote neuroregeneration

Neuronal polo-like kinase (nPLK) is an essential regular of cell cycle and differentiation in nervous system, and targeting nPLK has been established as a promising therapeutic strategy to treat neurological disorders and to promote neuroregeneration. The protein contains an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD) that are mutually inhibited by each other. Here, the intramolecular KD–PBD complex in nPLK was investigated at structural level via bioinformatics analysis, molecular dynamics (MD) simulation and binding affinity scoring. From the complex interface two regions representing separately two continuous peptide fragments in PBD domain were identified as the hot spots of KD–PBD interaction. Structural and energetic analysis suggested that one (PBD peptide 1) of the two peptides can bind tightly to a pocket nearby the active site of KD domain, which is thus potential as self-inhibitory peptide to target and suppress nPLK kinase activity. The knowledge harvesting from computational studies were then used to guide the structural optimization and mutation of PBD peptide 1. Consequently, two of three peptide mutants separately exhibited moderately and considerably increased affinity as compared to the native peptide. The computationally modeled complex structures of KD domain with these self-inhibitory peptides were also examined in detail to unravel the structural basis and energetic property of nPLK-peptide recognition and interaction.     

Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.     

Rational design and molecular diversity for the construction of anti-alpha-bungarotoxin antidotes with high affinity and in vivo efficiency

The structure of peptide p6.7, a mimotope of the nicotinic receptor ligand site that binds alpha-bungarotoxin and neutralizes its toxicity, was compared to that of the acetylcholine binding protein. The central loop of p6.7, when complexed with alpha-bungarotoxin, fits the structure of the acetylcholine binding protein (AChBP) ligand site, whereas peptide terminal residues seem to be less involved in toxin binding. The minimal binding sequence of p6.7 was confirmed experimentally by synthesis of progressively deleted peptides. Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new alpha-bungarotoxin binding peptides on the basis of their dissociation kinetic rate. The tetra-branched forms of the resulting high-affinity peptides were effective as antidotes in vivo at a significantly lower dose than the tetra-branched lead peptide.     

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.     

Computational analysis on the binding of epitope peptide to human leukocyte antigen class I molecule A*2402 subtype

Immunological response induced by small amino peptide has attracted much recent attention in the field of immunotherapy. Wilms' tumor (WT1) protein is one of the potent tumor antigens inducing immunological response in mouse and human, because WT1 is over expressed in many types of leukemia and various kinds of solid tumors. A 9-mer peptide encoded in WT1 protein (CMTWNQMNL; amino acid 235-243) is known to serve as antigenic peptide for human leukocyte antigen (HLA)-A*2402 molecule. It was reported that the replacement of the second amino residue, which is deeply responsible for the peptide binding to HLA, induced strong immunological response compared to the natural peptide. In this study, 19 kinds of single amino substitutions were introduced at position 2 of this 9-mer WT1 peptide. We performed molecular dynamics simulation on the complex of each of WT1 epitope peptides and HLA-β2 micro globulin (β2m) molecule, and subsequently estimated the binding affinity using molecular mechanics/generalized-Born surface area method combined with normal mode analysis. Our computation indicated that the peptide containing M2Y or M2W mutation showed high binding affinity to the HLA-β2m molecule as well as the natural peptide. We have also examined the role of the residue at position 2 in peptide binding to HLA-β2m. The calculation showed that van der Waals interaction between the side chain of the residue at position 2 and hydrophobic residues inside B-pocket of HLA are important. These findings will be helpful to search other potent peptides that will enhance strong immunological response specific to HLA-A*2402 molecule.     

Site-selective metal binding by designed alpha-helical peptides

It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)4G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of 113Cd, 1H NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.     

Lipid-protein interactions in the membrane: studies with model peptides

We have used fluorescence quenching of tryptophan-containing trans-membrane peptides by bromine-containing phospholipids to study the specificity of peptide-lipid interactions. We have synthesized peptides Ac-K2GLm WLnK2A-amide where m = 7 and n = 9 (L16) and m = 10 and n = 12 (L22). Binding constants of L22 for dioleoylphosphatidylserine [di(C18 : 1)PS] or dioleoylphosphatidic acid [di(C18 : 1)PA] relative to dieoleoylphosphatidylcholine [di(C18 : 1)PC] were close to 1. However, for L16, whilst the bulk of the di(C18 : 1)PA molecules bound with a binding constant relative to di(C18 : 1)PC close to 1, a small number of di(C18 : 1)PA molecules bound much more strongly. Assuming just one high affinity binding site on L16 for anionic lipid, the affinity of the site for di(C18 : 1)PS was calculated to be ca. 8 times that for di (C18 : 1)PC. The relative binding constant was little affected by ionic strength and close contact between the anionic headgroup of di(C18 : 1)PS and a lysine residue on the peptide was suggested. The relative binding constant for di(C18 : 1)PS at this high affinity site was less than for di(C18 : 1)PA. Cholesterol interacts with L22 with an affinity about 0.7 of that of di(C18 : 1)PC. The structure of the peptide itself is important. The peptide Ac-KKGYL6WL8YKKA-amide (Y2L14) incorporated into bilayers of dinervonylphosphatidylcholine [di(C24 : 1)PC] whereas L16 did not incorporate into this lipid. It is suggested that thinning of a lipid bilayer around a peptide to give optimal hydrophobic matching is less energetically unfavourable when a Tyr residue is located in the lipid/water interfacial region.     

Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry

Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in Cpe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the Cpe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased approximately 7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at approximately 10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides.     

Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase

We applied peptide array methylation to determine an optimized target sequence for the SET7/9 (KMT7) protein lysine methyltransferase. Based on this, we identified 91 new peptide substrates from human proteins, many of them better than known substrates. We confirmed methylation of corresponding protein domains in?vitro and in?vivo with a high success rate for strongly methylated peptides and showed methylation of nine nonhistone proteins (AKA6, CENPC1, MeCP2, MINT, PPARBP, ZDH8, Cullin1, IRF1, and [weakly] TTK) and of H2A and H2B, which more than doubles the number of known SET7/9 targets. SET7/9 is inhibited by phosphorylation of histone and nonhistone substrate proteins. One lysine in the MINT protein is dimethylated in?vitro and in?vivo demonstrating that the product pattern created by SET7/9 depends on the amino acid sequence context of the target site.     

Truncation,modification, and optimization of MIG6segment 2 peptide to target lung cancer-related EGFR

Human epidermal growth factor receptor (EGFR) plays a central role in the pathological progression and metastasis of lung cancer; the development and clinical application of therapeutic agents that target the receptor provide important insights for new lung cancer therapies. The tumor-suppressor protein MIG6 is a negative regulator of EGFR, which can bind at the activation interface of asymmetric dimer of EGFR kinase domains to disrupt dimerization and then inactivate the kinase (Zhang X. et al. Nature 2007, 450: 741–744). The protein adopts two separated segments, i.e. MIG6^{segment 1} and MIG6^{segment 2}, to directly interact with EGFR. Here, computational modeling and analysis of the intermolecular interaction between EGFR kinase domain and MIG6^{segment 2} peptide revealed that the peptide is folded into a two-stranded β-sheet composed of β-strand 1 and β-strand 2; only the β-strand 2 can directly interact with EGFR activation loop, while leaving β-strand 1 apart from the kinase. A C-terminal island within the β-strand 2 is primarily responsible for peptide binding, which was truncated from the MIG6^{segment 2} and exhibited weak affinity to EGFR kinase domain. Structural and energetic analysis suggested that phosphorylation at residues Tyr394 and Tyr395 of truncated peptide can considerably improve EGFR affinity, and mutation of other residues can further optimize the peptide binding capability. Subsequently, three derivative versions of the truncated peptide, including phosphorylated and dephosphorylated peptides as well as a double-point mutant were synthesized and purified, and their affinities to the recombinant protein of human EGFR kinase domain were determined by fluorescence anisotropy titration. As expected theoretically, the dephosphorylated peptide has no observable binding to the kinase, and phosphorylation and mutation can confer low and moderate affinities to the peptide, respectively, suggesting a good consistence between the computational analysis and experimental assay.     

Peptide-Hypervalent Iodine Reagent Chimeras: Enabling Peptide Functionalization and Macrocyclization**

Herein, we report a novel strategy for the modification of peptides based on the introduction of highly reactive hypervalent iodine reagents—ethynylbenziodoxolones (EBXs)—onto peptides. These peptide-EBXs can be readily accessed, by both solution- and solid-phase peptide synthesis (SPPS). They can be used to couple the peptide to other peptides or a protein through reaction with Cys, leading to thioalkynes in organic solvents and hypervalent iodine adducts in water buffer. Furthermore, a photocatalytic decarboxylative coupling to the C-terminus of peptides was developed using an organic dye and was also successful in an intramolecular fashion, leading to macrocyclic peptides with unprecedented crosslinking. A rigid linear aryl alkyne linker was essential to achieve high affinity for Keap1 at the Nrf2 binding site with potential protein-protein interaction inhibition.     

Identification of Chemokine Ligands by Biochemical Fragmentation and Simulated Peptide Evolution

Short linear peptides can overcome certain limitations of small molecules for targeting protein–protein interactions (PPIs). Herein, the interaction between the human chemokine CCL19 with chemokine receptor CCR7 was investigated to obtain receptor‐derived CCL19‐binding peptides. After identifying a linear binding site of CCR7, five hexapeptides binding to CCL19 in the low micromolar to nanomolar range were designed, guided by pharmacophore and lipophilicity screening of computationally generated peptide libraries. The results corroborate the applicability of the computational approach and the chosen selection criteria to obtain short linear peptides mimicking a protein–protein interaction site.     

Specific binding of GM1-binding peptides to high-density GM1 in lipid membranes

The ganglioside Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1) is an important receptor. We have previously identified GM1-binding peptides based on affinity selection from a random peptide library. In the present study, we determined the amino acids essential for binding GM1 and investigated the specific interaction with GM1 in the lipid membrane. Arginines and aromatic amino acids in the consensus sequence (W/F)RxL(xP/Px)xFxx(Rx/xR)xP contributed to the ability of the peptides to bind GM1. The peptide p3, VWRLLAPPFSNRLLP, having the consensus sequence, showed high affinity for GM1 with a dissociation constant of 1.2 microM. Furthermore, the density-dependent binding of p3 was investigated using mixed monolayers of GM1 and Glcbeta1-1'Cer (GlcCer). p3 binds preferentially to high-density GM1, and its interaction with GM1 was found to be cooperative based on a Hill plot. These results indicated that a lateral assembly of GM1 molecules was required for the recognition of carbohydrates by p3. The GM1-binding peptide played a role as a unique anti-GM1 probe differing from the cholera toxin B subunit or antibodies.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.