Effect of covalent binding of aprotinin with a polysaccharide carrier on its inhibition of kallikrein from human plasma, porcine pancreatic kallikrein and trypsin

The inhibition of trypsin, human blood plasma kallikrein and porcine pancreatic kallikrein by aprotinin (native and immobilized on carboxymethyl ester of dextran) was investigated. The experimental values of Ki of native and immobilized aprotinin--enzyme complexes are equal to 0.037 and 0.045 nM for trypsin, 0.38 and 112.3 nM for pancreatic kallikrein and 34.4 and 454.5 nM for plasma kallikrein with N alpha-benzoyl-L-arginine ethyl ester as substrate, and to 82.6 and 231.7 nM for plasma kallikrein with a natural substrate--kininogen. These data suggest that covalent binding of aprotinin to the water-soluble polysaccharide carrier does not interfere with its interaction with trypsin, whereas the inhibition of kallikreins decreases, especially that of pancreatic kallikrein. The experimental results indicate the marked differences in the structure of the binding site of the active center (or its environment) of plasma and pancreatic kallikreins, on one hand, and trypsin, on the other, as well as the differences between the plasma and pancreatic kallikreins. A high requirement of kallikreins to the maintenance of the native conformation of aprotinin during immobilization is postulated.     

A strong homology exists between the active T-cell binding gp120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A

A homology has been found between an octapeptide involved in attachment of the human immunodeficiency virus to helper/inducer T cells and an octapeptide segment of bovine pancreatic ribonuclease A. This segment (residues 19-26) contains the sites for subtilisin cleavage of this enzyme into the S-peptide and S-protein. From the X-ray crystal structure of ribonuclease, this sequence is known to be exposed to solvent and interacts little with the rest of the protein. A structure for the human immunodeficiency virus attachment peptide can be deduced from this homology, as a well-defined structure has been determined for this sequence in ribonuclease. This can be readily accomplished using previously developed computer methods based upon conformational energy calculations. The calculated structure for human immunodeficiency virus peptide is identical to the ribonuclease segment (19-26) in backbone conformation. It is stabilized by internal interactions of nonpolar residues, and by exposure of polar hydroxyl groups. The results suggest that the T-cell human immunodeficiency virus receptor may be hydrophilic in nature and that conservation of the sequence in two presumably functionally unrelated proteins is related to the need for conservation of exposed structure.     

Enzymatic resynthesis of the "reactive site" bond in the modified aprotinin derivatives [seco-15/16]aprotinin and [Di-seco-15/16,39/40]aprotinin

On incubation of [di-seco-15/16,39/40]aprotinin with human plasmin, porcine pancreatic kallikrein or bovine or porcine trypsin in neutral or slightly alkaline solutions [seco-39/40]aprotinin is slowly formed with enzymatic resynthesis of the reactive-site bond 15/16. With chymotrypsin, however, further degradation of [di-seco-15/16,39/40]aprotinin takes place without enzymatic resynthesis. The apparent rate constants for the synthesis of [seco-39/40]aprotinin with kallikrein and trypsin have been determined and indicate that the bond-forming reaction is 10-200-fold slower with [di-seco-15/16,39/40]aprotinin than with [seco-15/16]aprotinin. The newly formed [seco-39/40]aprotinin has similar kinetic constants for the complexation with its cognate enzymes as aprotinin, indicating that any distortion of the secondary binding region due to cleavage of the Arg39-Ala40 bond does not seriously influence binding and affinities.     

Membrane glycoprotein synthesis: cleavage of the signal sequence in the absence of glycosylation

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly α-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme. Moreover, proton magnetic resonance spectroscopy shows that the peptide adopts a thermostable tertiary structure with characteristics typical of globular proteins. The results as a whole indicate that the activation segment of porcine pancreatic procarboxypeptidase A constitutes a folded structural domain.     

Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor: Crystallization,patterson search,structure determination,refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibitor complex

The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P4₁2₁2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.The root-mean-square deviation, including all main-chain atoms, is 0·45 Å and 0·65 Å for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 Å away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 Å hydrogen bond with His57 NE.The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al., 1983).     

Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4₁2₁2 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined.A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen &; Bode. 1983).The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen &; Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.     

Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and those of the peptide, as well as chemical shifts of peptide resonances induced by the binding of MgATP, are consistent with the previously proposed binding site for MgATP on adenylate kinase.     

Proton NMR and CD solution conformation determination and opioid receptor binding studies of a dynorphin A(1-17) model peptide

The opioid peptide dynorphin A(1-17) contains a peptide segment in residues 7-15 with the potential to form an amphiphilic beta-strand. This amphiphilic structure may, like the amphiphilic alpha-helices found in many other peptide hormones, be an important determinant of its interactions with membranes and receptors. In order to investigate and characterize these interactions, we have synthesized a 17-residue dynorphin analogue (YGGFLKKVKPKVKVKSS) that incorporates a peptide model of this amphiphilic secondary structure with minimized homology (25%) relative to the native sequence. This peptide exhibits the full biological potency of dynorphin in assays of kappa-opioid receptor binding, and is more selective for this type of opioid receptor than the natural peptide. The conformation of the model peptide in aqueous solution has been investigated in detail by NMR spectroscopy. The values of the NH-CH alpha coupling constants together with rotating frame NOEs indicate the presence of an amphiphilic structure together with some beta-strand structure in residues 7-15, and demonstrate that a peptide model that stabilizes this structure in aqueous solution and enhances kappa-opioid receptor selectivity can be successfully designed using using alternating lysine and valine residues.     

Solution structure of monomeric peptide YY supports the functional significance of the PP-fold

Peptide YY (PYY) belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic peptide (PP) that regulate numerous functions through both central and peripheral receptors. The solution structure of these peptides is hypothesized to be critically important in receptor selectivity and activation, based on prior demonstration of a stable tertiary conformation of PP called the "PP-fold". Circular dichroism (CD) spectra show a pH-dependent structural transition in the pH range 3-4. Thus we describe the tertiary structure of porcine PYY in water at pH 5.5, 25 degrees C, and 150 mM NaCl, as determined from 2D (1)H NMR data recorded at 500 MHz. A constraint set consisting of 396 interproton distances from NOE data was used as input for distance geometry, simulated annealing, and restrained energy minimization calculations in X-PLOR. The RMSDs of the 20 X-PLOR-generated structures were 0.71 +/- 0.14 and 1.16 +/- 0.17 A, respectively, for backbone and heavy atom overlays of residues 1-34. The resulting structure consists of two C-terminal helical segments from residues 17 to 22 and 25 to 33 separated by a kink at residues 23, 24, and 25, a turn centered around residues 12-14, and the N-terminus folded near residues 30 and 31. The well-defined portions of the PYY structure reported here bear a marked similarity to the structure of PP. Our findings strongly support the importance of the stable folded structure of this family of peptides for binding and activation of Y receptor subtypes.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

1H NMR studies of bovine and porcine phospholipase A2: assignment of aromatic resonances and evidence for a conformational equilibrium in solution

Bovine and porcine pancreatic phospholipases A2, and porcine isophospholipase A2, have been investigated by one- and two-dimensional 1H NMR spectroscopy. Resonances have been assigned for 20-26 residues in each enzyme, including all the aromatic residues, by a strategy based on the semiquantitative comparison of proximity relationships deduced from NOE experiments with those seen in the crystal structure NOE experiments indicate that the loop comprising residues 59-70, which has a different conformation in the crystal structures of the bovine and porcine enzymes, has the same conformation in these two enzymes in solution. Selective changes in the line width of a limited number of resonances as a function of pH, temperature, and calcium concentration provide evidence for a local conformational equilibrium. This equilibrium involves a limited region of the protein structure around residues 25, 41, 106, and 111; it has been identified in the bovine enzyme and porcine isoenzyme but is not apparent in the porcine enzyme.     

The structure of the amino terminal transforming segment of the p21 protein, Tyr4-Thr20 (with Asp12), by two-dimensional NMR

The structure of a peptide from the transforming region (residues 4-20) of the p21 protein has been determined using two-dimensional NMR. In the normal protein, this segment contains a Gly residue at the critical 12 position; any substitution, other than Pro, at this position results in a transforming protein. Previously performed energy calculations indicated that this peptide segment is a structured one. In this study we find that the Asp12 containing peptide has a surprisingly well-defined structure in solution which has more similarity to the GDP-binding loop region in EF-tu than to that in p21.     

Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis

A new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a step-wise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.     

胰岛素折叠、结合与稳定性的核磁共振研究(英)

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and bexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure“, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short a-helix at C-terminal of A chain linked. The “super-secondary structure“ might be the “folding nucleus“ in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C. elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin superfamily adopt the “insulin fold“ mostly. The structural study of insulin-insulin receptor complex, that of C elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.     

Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure

The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.     

Design of a small peptide-based proteinase inhibitor by modeling the active-site region of barley chymotrypsin inhibitor 2

A synthetic peptide-based proteinase inhibitor was constructed by modeling the regions responsible for inhibition in barley chymotrypsin inhibitor 2 (CI-2). The 18-residue peptide was designed by molecular modeling, based on the crystal structure of CI-2. The amino acid sequences that interact with the proteinase were preserved, as well as residues that maintain the structure of the inhibitory loop. A disulfide bridge was introduced to force the peptide to adopt a cyclic structure. Kinetic studies on binding of the cyclic peptide to subtilisin BPN', subtilisin Carlsberg, chymotrypsin, and pancreatic elastase show that the cyclic peptide retains both the inhibition properties, the kinetic mechanism, and the specificity of the original protein inhibitor. Formation of a cyclic structure was found to be essential, and activity was abolished by reduction of the disulfide. As with CI-2, tightest binding is found to subtilisin BPN', where the Ki value for the cyclic peptide was 28 x 10(-12) M, compared with 29 x 10(-12)M for CI-2 under identical conditions. This remarkable result shows that it is possible to use a short synthetic peptide to model the molecular recognition properties of the intact protein, in this case obtaining full functionality with just 18 residues instead of 83 for CI-2.     

Structure and function of the proline-rich region of myelin basic protein

Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.     

Identification and structure characterization of a Cdk inhibitory peptide derived from neuronal-specific Cdk5 activator

The activation of cyclin-dependent kinase 5 (Cdk5) depends on the binding of its neuronal specific activator Nck5a. The minimal activation domain of Nck5a is located in the region of amino acid residues 150 to 291 (Tang, D., Chun, A. C. S., Zhang, M., and Wang, J. H. (1997) J. Biol. Chem. 272, 12318-12327). In this work we show that a 29-residue peptide, denoted as the alphaN peptide, encompassing amino acid residues Gln145 to Asp173 of Nck5a is capable of binding Cdk5 to result in kinase inhibition. This peptide also inhibits an active phospho-Cdk2-cyclin A complex, with a similar potency. Direct competition experiments have shown that this inhibitory peptide does not compete with Nck5a or cyclin A for Cdk5 or Cdk2, respectively. Steady state kinetic analysis has indicated that the alphaN peptide acts as a non-competitive inhibitor of Cdk5. Nck5a complex with respect to the peptide substrate. To understand the molecular basis of kinase inhibition by the peptide, we determined the structure of the peptide in solution by circular dichroism and two-dimensional 1H NMR spectroscopy. The peptide adopts an amphipathic alpha-helical structure from residues Ser149 to Arg162 which can be further stabilized by the helix-stabilizing solvent trifluoroethanol. The hydrophobic face of the helix is likely to be the kinase binding surface.     

Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.