Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.     

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.     

Use of fragments of hirudin to investigate thrombin-hirudin interaction

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.     

Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site.

A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA. It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent. No smaller resistant fragments were produced. 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association. Under milder trypsin digestion conditions three resistant fragments were produced from the free protein. The largest corresponded to that isolated from the complex. The smaller ones were trimmed slightly further at both N- and C-terminal ends. These smaller fragments did not reassociate with 5S RNA. It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction. The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA.     

DNA binding alters the protease susceptibility of the p50 subunit of NF-kappa B.

The subdomain structure of the p50 subunit of NF-kappa B (amino acids 35-381) was investigated by partial proteolysis of the native protein. Trypsin cleaves p50 at a limited number of sites with an initial cleavage at low trypsin concentration occurring after R362 and a second cleavage taking place at higher trypsin concentration after K77. The cleavage after R362 does not alter the DNA binding characteristics of p50 but removes the nuclear localisation signal indicating that this region occupies a highly exposed position on the surface of the protein. The second cleavage after K77 generates a protein that although dimeric is incapable of binding DNA, thus emphasising the importance of residues 35-77 in DNA recognition. However p50 dimers containing one molecule cleaved after K77 and one molecule with this region intact are capable of binding DNA. When very high concentrations of trypsin are employed p50 is completely degraded. However if p50 is bound tightly to DNA containing its specific recognition site prior to trypsin addition the cleavage after K77 is almost completely blocked and the protein becomes highly resistant to proteolysis. These data suggest that bound DNA may mask critical trypsin cleavage sites or that DNA binding is accompanied by a conformational change in protein structure that renders the protein resistant to proteolysis.     

Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sake L45.

Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ion exchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purification resulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled the determination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocin S contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine, valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that the N-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence of the 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe- Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentified residues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique when compared to the SWISS-PROT data bank.     

Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.     

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The properties of the N-terminal and C-terminal halves of histone H1.

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1--106 and 107--C-terminal. These were studied by proton magnetic resonance and circular dichroism. The N-terminal fragment exhibited some salt-induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C-terminal fragments show very little salt-induced structure. The N-terminal fragments binds to DNA very weakly, but the C-terminal fragment binds as strongly as the whole molecule. In the C-terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 - DNA complexes, and it is suggested that there may be a connection between these phenomena.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.     

Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly

Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Changes at the N-terminus of human brain creatine kinase during a transition between inactive folding intermediate and active enzyme.

CK-STAR, a monoclonal antibody against human brain creatine kinase (CK), can be shown by chemical cleavage mapping and peptide synthesis to recognize an epitope at the free N-terminus of the enzyme. The epitope could be largely reproduced by a synthetic peptide based on the first 18 amino acids and could be partly formed by the first 11 amino acids. The antibody did not bind to native CK, but it did bind to CK in various partially denatured forms and to an enzymically inactive intermediate in the refolding process. Competitive binding studies have shown that the N-terminal conformations of both the refolding intermediate and the free peptide resemble that of CK partially denatured by attachment to plastic. The results suggest that the final stages of CK refolding and reactivation involve a structural change at the N-terminus or its interaction with some other part of the CK molecule, thus masking the CK-STAR epitope.     

Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sake L45

Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ion exchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purification resulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled the determination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocin S contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine, valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that the N-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence of the 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe- Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentified residues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique when compared to the SWISS-PROT data bank.     

A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.     

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site.

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.     

The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L). Identification of the trypsin-reactive site, partial-amino-acid sequence and further physico-chemical properties of the major inhibitor.

The two principal isoinhibitors P-5 and P-6 isolated earlier from the seeds of chick peas (Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix-bound trypsin are shown to be the virgin and trypsin-modified forms of the same inhibitor. The virgin inhibitor P-5 consists of a single peptide chain of 66 amino acid residues cross-linked by seven disulfide bridges. Upon interaction with the matrix-bound trypsin under the conditions used the virgin inhibitor P-5 is about 50% converted to P-6 by cleavage of a single Lys-Ser linkage at position 14-15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1-14 and 15-66 separate readily by molecular-sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence 1-25 was established by Edman degradation.