Conformation-based design of two cyclic physalaemin analogues

Two new cyclic analogues of physalaemin were designed on the basis of the conformation found in DMSO solution. Glp-Ala-cyclo(-Asp-Pro-Asn-Lys-)-Phe-Tyr-Gly-Leu-Met-NH2 (1) was synthesized by cyclization of physalaemin. In 2 the Asp residue was replaced by Glu. The linear analogue of 2 was synthesized by the solid phase method and subsequently cyclized. Two-dimensional nmr methods were employed to assign the proton and carbon resonances. Proton-proton distances were extracted from rotating frame nuclear Overhauser effect spectra and used as restraints in the molecular dynamics calculations. Analogue 1 was found to have a similar conformation as physalaemin, whereas 2 did not form intramolecular hydrogen bonds. The pharmacological evaluation revealed that both peptides have similar potencies as physalaemin in the dog carotid artery (NK-1 receptor). Therefore, the charged side chains of physalaemin appear not essential for NK-1 activation. However, the other tachykinin receptors show good sensitivity to the cyclic peptides. It is concluded that the replacement of a salt bridge by an amide bond connecting the side chains of natural residues might provide useful information about the biological significance of some charged side chains of neurokinins.     

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k _cat/K _m), half-lives (t _1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.     

Electrostatic interactions in wild-type and mutant recombinant human myoglobins

Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.     

Conformational analogy between substance P and physalaemin

The three-dimensional structure of physalaemin, pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2, has been studied by one- and two-dimensional 500 MHz NMR spectroscopies in two solvents: methanol and dimethyl sulfoxide. As previously observed for substance P in methanol, the core of physalaemin 4----8 is folded into an helical conformation. This structure is stabilized by the presence of a salt bridge between Asp-3 and Lys-6 in both solvents. The only differences observed reside in the N-terminal sequences; the N-terminal tripeptide of substance is flexible whereas that of physalaemin is in an extended conformation.     

Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins: solution structures of mouse CRYPTDIN-4 and (E15D)-CRYPTDIN-4

alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.     

Solution structure of contryphan-R, a naturally occurring disulfide-bridged octapeptide containing D-tryptophan: comparison with protein loops.

Contryphan-R is a disulfide-constrained octapeptide containing a D-tryptophan that was isolated recently from venom of the cone shell Conus radiatus. The polypeptide is present in two forms in solution due to cis-trans isomerization at hydroxyproline 3. The solution structure of the major form of this unusual polypeptide, determined from NMR data, consists of a well-defined fold containing a non-hydrogen-bonded chain reversal from Gly1 to Glu5, which includes a cis-hydroxyproline and a D-Trp, and a type I beta-turn from Glu5 to Cys8. The presence of a putative salt bridge between the Glu5 carboxyl group and the N-terminal ammonium group is investigated by using various solvation models during energy minimization and is compared with the results of a pH titration. A comparison of the structure of contryphan-R with other cyclic peptide structures highlights some of the key structural determinants of these peptides and suggests that the contryphan-R fold could be exploited as a scaffold onto which unrelated protein binding surfaces could be grafted. Comparison with small disulfide-bridged loops in larger proteins shows that contryphan-R is similar to a commonly occurring loop structure found in proteins.     

Conformational analysis of the tachykinins in solution: substance P and physalaemin.

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.     

Contribution of surface salt bridges to protein stability

The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1. 5 kcal mol(-)(1). The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.     

Solution structure of the cyclic peptide contryphan-Vn, a Ca2+-dependent K+ channel modulator

The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S-S bridge and containing a D-tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV beta-turn from Gly 1 to Lys 6 and a type I beta-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines--the Pro4--is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D-Trp and the two Pro residues.     

High performance screening, structural and molecular dynamics analysis to identify H1 inhibitors from TCM Database@Taiwan

New-type oseltamivir-resistant H1N1 influenza viruses have been a major threat to human health since the 2009 flu pandemic. To resolve the drug resistance issue, we aimed to identify a new type of inhibitors against H1 from traditional Chinese medicine (TCM) by employing the world's largest TCM database () for virtual screening and molecular dynamics (MD). From the virtual screening results, sodium (+)-isolaricireinol-2 alpha-sulfate, sodium 3,4-dihydroxy-5-methoxybenzoic acid methyl ester-4-sulfate, sodium (E)-7-hydroxy-1,7-bis(4-hydroxyphenyl)hept-5-ene-3S-sulfonate, and 3-methoxytyramine-betaxanthin were identified as potential drug-like compounds. MD simulation of the binding poses with the key residues Asp103 and Glu83, as well as other binding site residues, identified higher numbers of hydrogen bonds than N-Acetyl-D-Glucosamine (NAG), the natural ligand of the esterase domain in H1. Ionic bonds, salt bridges, and electrostatic energy also contribute to binding stability. Key binding residues include Lys71, Glu83, Asp103, and Arg238. Structural moieties promoting H-bond or salt bridge formations at these locations greatly contribute to a stable ligand-protein complex. An available sodium atom for ionic interactions with Asp103 can further stabilize the ligands. Based on virtual screening, MD simulation, and interaction energy evaluation, TCM candidates demonstrate good potential as novel H1 inhibitors. In addition, the identified stabilizing features can provide insights for designing highly stable H1 inhibitors.     

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

Abstract

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of β-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Effects of N-Acetyl Aspartate, Aspartate, and Glutamate on cAMP and cGMP Levels in Developing Rat Cerebral Cortex

N-Acetyl-aspartate (N-Ac-Asp) incubated with minced cerebral cortex caused a dose-dependent increase in the levels of cAMP and cGMP. This effect was followed during postnatal development. N-Ac-Asp elicits the greatest increase in cAMP in 5-day-old and in cGMP in 40-day-old rats. The levels of cyclic AMP were always higher than those of cGMP. We also studied the effects of L-aspartate (Asp) and L-glutamate (Glu) on the levels of cyclic nucleotides in the cerebral cortex minces of rats different ages, and observed that both amino acids produced the maximum increase in cAMP at 10 days, whereas in the case of cGMP the maximal effect of Asp occurs earlier than 20 days and of Glu after 40 days. In the adult rat, the N-Ac-Asp effect on cAMP was greater than that produced by either Asp or Glu, whereas the levels of cGMP were similarly affected by all three. The data show a peak response of cAMP and cGMP to N-Ac-Asp, Asp, and Glu during cortical maturation. Because this response varies with postnatal time, N-Ac-Asp, and Glu may act upon different receptor sites.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

Salt bridge interactions: Stability of the ionic and neutral complexes in the gas phase,in solution,and in proteins

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen-bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley-Liss, Inc.     

Internal and environmental effects on folding and dimerisation of Alzheimer's β-amyloid peptide

Amyloid deposits are a hallmark of many diseases. In the case of Alzheimer's disease, a turn between 21Ala and 30Ala, stabilised by a salt bridge between 22Glu/23Asp and 28Lys, may nucleate folding and aggregation of the amyloid β (Aβ) peptide. In the present paper, we test this hypothesis by studying how salt bridge and turn formation vary with intrinsic and environmental changes, and how these changes affect folding and aggregation of the Aβ-peptide.     

Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors

Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.     

Surface salt bridges,double-mutant cycles,and protein stability: an experimental and computational analysis of the interaction of the Asp 23 side chain with the N-terminus of the N-terminal domain of the ribosomal protein l9

Experimental and theoretical double-mutant cycles have been used to investigate a salt bridge in the N-terminal domain of the protein L9. Aspartic acid 23 is the only acidic residue involved in a well-defined pairwise interaction, namely, a partially solvent-exposed salt bridge with the protonated N-terminus of the protein. Mutations were studied in which Asp 23 was substituted by alanine, asparagine, and nitrile alanine. Interactions with the N-terminus were probed by comparisons between proteins with a protonated and acetylated N-terminus. The mutants were all folded, and the structures were unchanged from wild type as judged by CD and 2-D NMR. The coupling free energy between the N-terminus and the side chain of Asp 23 measured through double-mutant cycle analysis was favorable and ranged from -0.7 to -1.7 kcal mol(-)(1), depending upon the set of mutants used. This relatively large coupling free energy for a surface salt bridge likely arises from geometric factors that reduce the entropy loss associated with salt-bridge formation and from structural relaxation in the mutants. Coupling free energies computed with continuum electrostatic calculations agreed well with the experimental values when full account was taken of all potential interactions, particularly those involving Asp 23 and the acetylated N-terminus as well as interactions with solvent. The measured and calculated coupling free energy decreased only slightly when the salt concentration was increased from 100 to 750 mM NaCl. The calculations suggest that the coupling free energy between D23 and the N-terminus measured through the experimental double-mutant cycle analysis is significantly smaller than the actual interaction free energy between the groups in the wild-type structure because of the inapplicability of assumptions frequently used to interpret double-mutant cycles.     

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp(90) residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.