Relaxation matrix refinement of the solution structure of squash trypsin inhibitor

The structure of the small squash trypsin inhibitor CMTI-I is refined by directly minimizing the difference between the observed two-dimensional nuclear Overhauser enhancement (NOE) intensities and those calculated by the full relaxation matrix approach. To achieve this, a term proportional to this difference was added to the potential energy function of the molecular dynamics program X-PLOR. Derivatives with respect to atomic co-ordinates are calculated analytically. Spin diffusion effects are thus accounted for fully during the refinement. Initial structures for the refinement were those determined recently by solution nuclear magnetic resonance using the isolated two-spin approximation to derive distance range estimates. The fits to the nuclear magnetic resonance data improve significantly with only small shifts in the refined structures during a few cycles of conjugate gradient minimization. However, larger changes (approximately 1 A) in the conformation occur during simulated annealing, which is accompanied by a further reduction of the difference between experimental and calculated two-dimensional NOE intensities. The refined structures are closer to the X-ray structure of the inhibitor complexed with trypsin than the initial structures. The root-mean-square difference for backbone atoms between the initial structures and the X-ray structure is 0.96 A, and that between the refined structures and the X-ray structure 0.61 A.     

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.     

Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor.

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.     

1H-n.m.r. studies of squash seed trypsin inhibitor

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.     

Solution structure of BSTI: a new trypsin inhibitor from skin secretions of Bombina bombina

The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using (1)H NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cys (4--38), Cys (13--34), Cys (17--30), Cys (21--60), and Cys (40--54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta-strands, arranged as two small antiparallel beta-sheets. The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21--34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.     

Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.     

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.     

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.     

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.     

Chemical synthesis of new trypsin, chymotrypsin and elastase inhibitors by amino-acid substitutions in a trypsin inhibitor from squash seeds (CMTI III)

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.     

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.     

1H 2D NMR and distance geometry study of the folding of Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family

The solution conformation of synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with 3 disulfide bridges, has been studied by 1H 2D NMR measurements. Secondary structure elements were determined: a miniantiparallel beta-sheet Met 7-Cys 9 and Gly 25-Cys 27, a beta-hairpin 20-28 with beta-turn 22-25, and two tight turns Asp 12-Cys 15 and Leu 16-Cys 19. A set of interproton distance restraints deduced from two-dimensional nuclear Overhauser enhancement spectra and 13 phi backbone torsion angles restraints were used as the basis of three-dimensional structure computations including disulfide bridges arrangement by using distance geometry calculations. Computations for the 15 possible S-S linkage combinations lead to the proposal of the array 2-19, 9-21, 15-27 as the most probable structure for EETI II.     

Primary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor.

A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).     

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.     

Crystal structure reveals basis for the inhibitor resistance of human brain trypsin

Severe neurodegradative brain diseases, like Alzheimer, are tightly linked with proteolytic activity in the human brain. Proteinases expressed in the brain, such as human trypsin IV, are likely to be involved in the pathomechanism of these diseases. The observation of amyloid formed in the brain of transgenic mice expressing human trypsin IV supports this hypothesis. Human trypsin IV is also resistant towards all studied naturally occurring polypeptide inhibitors. It has been postulated that the substitution of Gly193 to arginine is responsible for this inhibitor resistance. Here we report the X-ray structure of human trypsin IV in complex with the inhibitor benzamidine at 1.7 A resolution. The overall fold of human trypsin IV is similar to human trypsin I, with a root-mean square deviation of only 0.5 A for all C(alpha) positions. The crystal structure reveals the orientation of the side-chain of Arg193, which occupies an extended conformation and fills the S2' subsite. An analysis of surface electrostatic potentials shows an unusually strong clustering of positive charges around the primary specificity pocket, to which the side-chain of Arg193 also contributes. These unique features of the crystal structure provide a structural basis for the enhanced inhibitor resistance, and enhanced substrate restriction, of human trypsin IV.