Beta-turn Region Research Articles

Structural impact due to PPQEE deletion in multiple cancer associated protein - Integrin αV: An In silico exploration

A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin β3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount β-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.

A combined view of B-cell epitope features in antigens.

B-cell epitope mapping is a promising approach to identify therapeutics and vaccine candidates in antigenic proteins. We used MATLAB programming to view in combination different features such as beta turn region, surface accessibility, antigenicity and hydrophilicity in an antigen sequence to help predict a discontinuous, conformational B-cell epitope. We analyzed, grouped, compared, matched and superposed these features for a combined visualization using MATLAB programming for identifying and illustrating a potential B-cell epitope region in an antigen protein. This protocol finds application in the design and development of an effective B cell epitope candidate.

B and T Cell Epitope-Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets.

Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence-based and available three-dimensional structural analyses of each protein of EBV. Peptides '(79) VPSATKRWGFRSGVPP(94) ' from GP1 and '(515) LHYWTTQDEGAAIGLA(530) ' from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region (519) TTQDEG(524) that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm. Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, 'LANETTQAL' and 'FLYDRLAST' nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA-DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine.

Membranolytic antifungal activity of arenicin-1 requires the N-terminal tryptophan and the beta-turn arginine

To determine the structural requirements of arenicin-1 in exerting antifungal activity, a truncated peptide with an N-terminal deletion and a peptide with an Ala substitution for an Arg in the beta-turn region were characterised by comparison to arenicin-1. The antifungal activities of the analogues were 25-50% lower than arenicin-1. Trp fluorescence and circular dichroism spectroscopy showed that Trp in the N-terminus contributed to peptide penetration and Arg in the beta-turn to conformational transition. These results suggest that Trp in the N-terminus and Arg in the beta-turn play a pivotal role in the membrane-directed antifungal activity of arenicin-1.

Ramachandran Revisited. DFT Energy Surfaces of Diastereomeric Trialanine Peptides in the Gas Phase and Aqueous Solution

We report DFT calculations at the B3LYP/D95(d,p) level on the gas phase, aqueous solvation and solvated energies as functions of the central psi and phi dihedral angles (in steps of 5 degrees each) of acetyl-(L)Ala-(L)Ala-(L)Ala-NH(2) (3AL) and its diastereomer, acetyl-(L)Ala-(D)Ala-(L)Ala-NH(2) (3AD). In addition to structures without internal H-bonds (C(5) interactions are neglected), many (95) structures containing internal H-bonds were completely optimized. The only minima for non-H-bonding structures in the gas phase correspond to extended beta-strands for both diastereomers. Some (but not all) structures with internal H-bonds are more stable than those without them. The energy landscapes for the solvated species show multiple minima for the non-H-bonding species and a single minimum for the H-bonding species (3(10)-helix), suggesting that the equilibrium conformational mixture in water be composed of the extended beta-strand, polyproline II, 3(10)-helix, and alpha-helix-like (with no H-bonds) conformations which are all within about 1 kcal/mol of each other. Most H-bonding structures are destabilized relative to the non-H-bonding structures in aqueous solution, but some with large dipole moments are not. The large dipole moment of the alpha-helix-like conformation leads to its increased stability in water (vs the gas phase). Significant qualitative and quantitative differences are reported for the energy landscapes of the two diastereomers when one is compared with the mirror image of the other landscape (particularly in the beta-turn region), suggesting that the differences in the energies of the unfolded peptides need to be considered when considering the stabilities of folded peptides and proteins with single amino acid mutations.

Aromatic Residues Engineered into the β-Turn Nucleation Site of Ubiquitin Lead to a Complex Folding Landscape, Non-Native Side-Chain Interactions, and Kinetic Traps

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

Arginine Dynamics in a Membrane‐Bound Cationic Beta‐Hairpin Peptide from Solid‐State NMR

The site-specific motion of Arg residues in a membrane-bound disulfide-linked antimicrobial peptide, protegrin-1 (PG-1), was investigated by using magic-angle-spinning solid-state NMR spectroscopy to better understand the membrane insertion and lipid interaction of this cationic membrane-disruptive peptide. The C-H and N-H dipolar couplings and 13C chemical shift anisotropies were measured in the anionic POPE/POPG membrane, and were found to be reduced from the rigid-limit values by varying extents; this indicates the presence of segmental motion. An Arg residue at the beta-turn region of the peptide showed much weaker spin interactions, which indicates larger amplitudes of motion than an Arg residue in the beta-strand region of the peptide. This is consistent with the exposure of the beta turn to the membrane surface and the immersion of the beta strand in the hydrophobic middle of the membrane, and supports the previously proposed oligomerization of the peptide into beta barrels in the anionic membrane. The 13C T2 and 1H T(1rho) relaxation times indicate that the beta-turn backbone undergoes large-amplitude intermediate-timescale motion in the fluid phase of the membrane; this causes significant line broadening and loss of spectral intensity. This study illustrates the strong correlation between the dynamics and structure of membrane proteins, and the capability of solid-state NMR spectroscopy to provide detailed information on site-specific dynamics in complex membrane-protein assemblies.

Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling

Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel beta-strands connected by two overlapping type I beta-turns stabilized by the aspartate and threonine residues. The beta-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to Keap1 and for Keap1-mediated repression of Nrf2-dependent gene expression. Our results provide a detailed picture of how a BTB-Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.

Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness

Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all sweetness. The same pattern occurred again at the beta-turn region, where Glu41Lys gave the highest sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey sweet taste in primates.

Structures of neuropeptide gamma from goldfish and mammalian neuropeptide gamma, as determined by 1H NMR spectroscopy.

Neuropeptide gamma belongs to tachykinin families which have a common C-terminal amino acid sequence (Phe-X-Leu-Met-NH2) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide gamma (NPgamma) from gold fish (G-NPgamma) and mammalian NPgamma (M-NPgamma), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G-NPgamma has a alpha-helical conformation in the region of His12-Met21 and a short helix in the N-terminal region, and has a beta-turn from Arg9 to Arg11 in between. In aqueous TFE solution, M-NPgamma also has alpha-helical conformations both in the C-terminal region and the N-terminal region and a beta-turn from His9 to Arg11 in between. In SDS micelle, the structure of G-NPgamma contains a stable alpha-helix from His12 to Met21 and a beta-turn from Arg9 to Arg11, while M-NPgamma has a short helix from Ser16 to Met21. The region from His12 to Met21 corresponds to the amino acid sequence of neurokinin A. Neuropeptide gamma may act as a precursor of neurokinin A and the post-translational processing of this peptide involves the enzymatic attack of the basic beta-turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G-NPgamma induces the biological actions corresponding to those of substance P in mammalian system. The structures of G-NPgamma and M-NPgamma contain alpha-helical structures at the C-terminus and this helix seems to promote the affinity for NK1 and/or NK2 receptor.

Use of phage display to probe the evolution of binding specificity and affinity in integrins.

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.

Solution Structure of a Kunitz-type Chymotrypsin Inhibitor Isolated from the Elapid Snake Bungarus fasciatus

Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions.

Energy landscape of a peptide consisting of alpha-helix, 3(10)-helix, beta-turn, beta-hairpin, and other disordered conformations.

The energy landscape of a peptide [Ace-Lys-Gln-Cys-Arg-Glu-Arg-Ala-Nme] in explicit water was studied with a multicanonical molecular dynamics simulation, and the AMBER parm96 force field was used for the energy calculation. The peptide was taken from the recognition helix of the DNA-binding protein, c-MYB: A rugged energy landscape was obtained, in which the random-coil conformations were dominant at room temperature. The CD spectra of the synthesized peptide revealed that it is in the random state at room temperature. However, the 300 K canonical ensemble, Q(300K), contained alpha-helix, 3(10)-helix, beta-turn, and beta-hairpin structures with small but notable probabilities of existence. The complete alpha-helix, imperfect alpha-helix, and random-coil conformations were separated from one another in the conformational space. This means that the peptide must overcome energy barriers to form the alpha-helix. The overcoming process may correspond to the hydrogen-bond rearrangements from peptide-water to peptide-peptide interactions. The beta-turn, imperfect 3(10)-helix, and beta-hairpin structures, among which there are no energy barriers at 300 K, were embedded in the ensemble of the random-coil conformations. Two types of beta-hairpin with different beta-turn regions were observed in Q(300K). The two beta-hairpin structures may have different mechanisms for the beta-hairpin formation. The current study proposes a scheme that the random state of this peptide consists of both ordered and disordered conformations. In contrast, the energy landscape obtained from the parm94 force field was funnel like, in which the peptide formed the helical conformation at room temperature and random coil at high temperature.

The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method.

A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.

Study on the packing geometry, stoichiometry, and membrane interaction of three analogs related to a pore-forming small globular protein.

A de novo designed pore-forming small globular protein (SGP) with antitumor activity consists of four helices: 3 basic amphipathic helices composed of Leu and Lys surrounding a central hydrophobic helix composed of oligoalanine. These helices are connected by a beta-turn-forming sequence and two beta-turn-unfavorable ones (S. Lee, T. Kiyota, T. Kunitake, E. Matsumoto, S. Yamashita, K. Anzai, and G. Sugihara Biochemistry 1997, Vol. 36, pp. 3782-3791). In the present work, we designed and synthesized three new SGP analogs in order to study the stoichiometric packing geometry and stability of SGP. The replacement of alanines in the central helix of SGP with leucines (SGP-L), which make the helix much larger in size and more hydrophobic, resulted in an equilibrium of monomeric-trimeric structure. The replacement of some Lys residues by Glu residues in the hydrophilic regions of the amphipathic helices (SGP-E) led to a decrease in helical content and the formation of an equilibrium of monomeric-trimeric structure. The alteration of beta-turn regions with Gly residues, which makes these regions flexible (SGP-G), established an equilibrium of monomeric-dimeric states in buffer. The hydrophobic alpha-helix of SGP-L penetrated into the lipid bilayers in a manner that stabilized model membranes and biomembranes, whereas the central helices of SGP-G and -E destabilized them by forming channels. SGP and its analogs may be a useful model to study the role of the hydrophobic and hydrophilic regions in the formation of monomer-oligomer of proteins and to better understand the insertion of membrane targeting proteins into biomembranes.

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.

Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2.

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N-glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta-turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15-18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16-19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.

Topographical amino acid substitution in position 10 of glucagon leads to antagonists/partial agonists with greater binding differences.

The role of position 10 in the beta-turn region of glucagon was investigated by substituting chiral constrained amino acids and other modifications in the N-terminal region. A series of glucagon analogues have been designed and synthesized by incorporating beta-methylphenylalanine isomers (2S,3S, 2S,3R, 2R,3R, and 2R,3S) at position 10 in order to explore the structural and topographical requirements of the glucagon receptor, and, in addition, utilizing previous studies which indicated that antagonism could be enhanced by modifications (des-His1, Glu9) and a bulky group at position 5. The structures of the new analogues are as follows: [des-His1,-Tyr5,Glu9]glucagon-NH2 (II), [des-His1,Tyr5,Glu9,Phe10]glucagon-NH2 (III), [des-His1,Tyr5,Glu9,-Ala10]glucagon-NH2 (IV), [des-His1,Tyr5,Glu9,(2S,3R)-beta-MePhe10]glucagon-NH2 (V), [des-His1,-Tyr5,Glu9,(2S,3S)-beta-MePhe10]glucagon-NH2 (VI), [des-His1,Tyr5,Glu9,D-Tyr10]glucagon-NH2 (VII), [des-His1,Tyr5,Glu9,D-Phe10]glucagon-NH2 (VIII), [des-His1,Tyr5,Glu9,D-Ala10]glucagon-NH2 (IX), [des-His1,Tyr5,Glu9,(2R,3R)-beta-MePhe10]glucagon-NH2 (X), and [des-His1,Tyr5,Glu9,(2R,3S)-beta-MePhe10]glucagon-NH2 (XI). These analogues led to dramatically different changes in in vitro binding affinities for glucagon receptors. Their receptor binding potencies IC50 values (nM) are 2.3 (II), 4.1 (III), 395.0 (IV), 10.0 (V), 170.0 (VI), 74.0 (VII), 34.5 (VIII), 510.0 (IX), 120.0 (X), and 180.0 (XI). Analogues II, III, V, VI, and XI were found to be weak partial agonists/partial antagonists with maximum stimulation between 5%-9%, while the other compounds (IV and VII-X) were antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10(-5) M. In competition experiments, all of the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 6.60 (II), 6.85 (III), 6.20 (IV), 6.20 (V), 6.10 (VI), 6.50 (VII), 6.20 (VIII), 5.85 (IX), 6.20 (X), and 6.00 (XI). Putative topographical requirements of the glucagon receptor for the aromatic side chain conformation in position 10 of glucagon antagonists are discussed.

Isoform-specific Differences in the Potencies of Murine Protein Kinase Inhibitors Are Due to Nonconserved Amino-terminal Residues

We provide here a detailed characterization of two isoforms of the protein kinase inhibitor (PKI) protein of cAMP-dependent protein kinase that have dramatically different inhibition constants. Murine PKI beta 1 possesses a 32-fold higher Ki than murine PKI alpha as determined by Henderson analysis. This finding led to the investigation of C subunit.PKI interactions involving nonconserved regions in the carboxyl and amino termini of murine PKI alpha and PKI beta 1. Chimeric cDNAs coding for amino acid sequences from both PKI isoforms were constructed and expressed in bacteria. Surprisingly, exchanging the carboxyl-terminal two-thirds of PKI alpha and PKI beta 1 has relatively little effect on the inhibition constants of the two isoforms. Similarly, introducing amino acid residues corresponding to a beta-turn region of PKI alpha into PKI beta 1 fails to lower PKI beta 1 inhibition constants. However, introducing the amino-terminal alpha-helical region of PKI alpha into PKI beta 1 reduces the Ki and IC50 of PKI beta 1 to values identical with full length PKI alpha. Site-directed mutagenesis of specific residues within this region implicates the presence of a tyrosine at position 7 in PKI alpha as a major contributor to its enhanced inhibitory potency. The results of this study suggest that variations in C subunit.PKI interactions within an amino-terminal alpha-helix provide a major mechanism for altering the inhibitory properties of PKI isoforms.

Small Peptide Mimics of Nerve Growth Factor Bind TrkA Receptors and Affect Biological Responses

Small monomeric cyclic analogs that mimic the beta-turn regions of nerve growth factor (NGF) were designed and synthesized. Potent competitive antagonists were derived from the NGF beta-turn C-D, which inhibited [125I] NGF binding to TrkA receptors and specifically inhibited optimal NGF-mediated neurite outgrowth in PC12 cells. The cyclic beta-turn A'-A" analog also inhibited NGF binding to TrkA receptors but with lower potency. These data indicate that beta-turns C-D and A'-A" are critical for TrkA binding and may confer neurotrophin receptor specificity. Furthermore, structural requirements for binding are absolute, because unconstrained analogs derived from the same regions had no effect. Compounds that mimic NGF will be useful in deciphering the interactions of NGF and its receptors and in rational drug design.