Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.     

Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase

The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.     

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands

PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate- binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K(+) channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.     

Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.     

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Primary sequence of the beta-chain of Badger haemoglobin.

Badger (Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The beta-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin beta-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro5, Ser/Ala13, Tyr/Phe41, Asp/Glu43, Ser/Ala70, Glu/Asp73, Lys/Ala76, Asn/His77, Lys/Thr87, Lys/Arg104 and Gln/Pro125. A comparison with other haemoglobin beta-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of beta-chain of known structure in the order of Carnivores.     

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.     

Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.     

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)₄-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (K_m = 15 μM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the K_m 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.     

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.