Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Crystal structure and function of C-terminal Sau3AI domain

Sau3AI is a type II restriction enzyme that recognizes the 5'-GATC-3' sequence in double-strand DNA and cleaves at 5' to the G residue. The C-terminal domain of Sau3AI (Sau3AI-C), which contains amino acids from 233 to 489, was crystallized and its structure was solved by using the Multi-wavelength Anomalous Diffraction method. The Sau3AI-C structure at 1.9 A resolution is similar to the structure of MutH, a DNA mismatch repair protein that shares high sequence similarity with the N-terminal Sau3AI domain. The functional analysis shows that Sau3AI-C can bind DNA with one recognition sequence but has no cleavage activity. These results indicate that Sau3AI is a pseudo-dimer belonging to the type IIe restriction enzymes and the Sau3AI-C is the allosteric effector domain that assists DNA binding and cleavage.     

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.     

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

Structure of the prion Ure2p in protein fibrils assembled in vitro

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers. Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.     

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.     

A novel beta-defensin structure: a potential strategy of big defensin for overcoming resistance by Gram-positive bacteria

Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a beta-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.     

NMR mapping of copper binding sites in alpha-synuclein

Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.     

NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3.

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-alpha-D-GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N-terminal PDT segment whereas the C-terminal RP segment has fewer interactions with the protein. In contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.     

Modular architecture of the hexameric human mitochondrial DNA helicase

We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. This novel helicase shares substantial amino acid sequence and functional similarities with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial DNA helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-terminal sequencing reveals distinct N and C-terminal polypeptides that represent minimal structural domains. Physical analysis of the proteolytic products defines the region required to maintain oligomeric structure to reside within amino acid residues approximately 405-590. Truncations of the N and C termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is functional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and secondary structural alignments combined with biochemical data suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. The hexameric conformation and modular architecture revealed in our study document that the mitochondrial DNA helicase and bacteriophage T7 primase-helicase share physical features. Our findings place the mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases.     

Functional characterization of the phosphorelay protein Mpr1p from Schizosaccharomyces pombe

Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.     

The receptor binding domain of apolipoprotein E is responsible for its antioxidant activity

Apolipoprotein E (apoE) is a 34-kDa lipid-associated protein present in plasma and in the central nervous system. Previous studies have demonstrated that apoE has multiple functions, including the ability to transport lipids, regulate cell homeostasis, and inhibit lipid oxidation. The lipid binding domain of apoE has been localized to the carboxyl-terminal domain, whereas a cluster of basic amino acid residues within the N-terminal domain is responsible for its receptor binding activity. This study was undertaken to identify the domain in apoE responsible for its antioxidant activity. Results showed that apoE inhibits Cu(2+)-induced LDL oxidation by delaying conjugated diene formation in a concentration-dependent manner. Reductive methylation of lysine residues or cyclohexanedione modification of arginine residues in apoE abolished its ability to inhibit LDL oxidation. Additional studies showed that a 22-kDa peptide containing the N-terminal domain of apoE3 was more effective than a similar peptide with the apoE4 sequence in inhibiting Cu(2+)-induced LDL oxidation. In contrast, the 10-kDa peptide that contains the C-terminal domain of apoE was ineffective. Inhibition of Cu(2+)-induced LDL oxidation can also be accomplished with a peptide containing either a single sequence or a tandem repeat sequence of the receptor binding domain (residues 141-155) of apoE. Taken together, these results localized the antioxidant domain of apoE to its receptor binding domain and the basic amino acids in this domain are important for its antioxidant activity.     

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.     

Terminal amino acid sequences and proteolytic cleavage sites of mouse mammary tumor virus env gene products

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.     

On the structure of proteoglycan core

Core protein from bovine nasal proteoglycan has been obtained by cyanogen bromide cleavage and by removal of most of the glycosaminoglycan side chains by hydrogen fluoride treatment. Amino acid analysis of the cyanogen bromide fragment shows it to consist mainly of proline, serine, glycine and glutamic acid (glutamine). End-group analyses of the fragment and HF stripped core reveal and N-terminal residue to be valine in each case. The stripped core has been subjected to sequencing and some sequential information is presented. Based upon the amino acid analysis, sequence information and other properties, conformation analysis indicates that the most likely conformation is that of a flexible extended chain containing β-turns. The existence of a common N-terminal residue indicates that it is the C-terminal region which lies in the region of the hyaluronic acid backbone in intact proteoglycan. Furthermore, enzymatic cleavage of core protein which occurs in proteoglycan turnover, aging and degenerative diseases, probably does not occur by a stepwise cleavage from the N terminus of proteoglycan but by a more drastic degradation process.     

The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains

A biochemical approach to identify proteins with high affinity for choline-containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC ) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted M r of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was purified from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 30 degrees C. The DNA region encoding the 253 C-terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but significant, choline-independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self-alignment of the N-terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline-binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline-binding domain and the catalytic domain are located, respectively, at the N-terminal and the C-terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline-binding proteins to fuse separated catalytic and substrate-binding domains and create new and functional mature proteins.     

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)