Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution

The crystal structure of recombinant human interleukin-1 beta (IL-1 beta) has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel beta-strands and no alpha-helix. The single 153-residue polypeptide chain is folded into a six-stranded beta-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the beta-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of beta-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1 beta receptors.     

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 Å) and biochemical basis

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu → Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8 × 10¹⁰ M^?1 which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 Å) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.     

The three-dimensional structure of interleukin-1 beta

The three-dimensional structure of human recombinant interleukin-1 beta has been determined at 0.24 nm resolution by X-ray crystallographic techniques. The partially refined model has a crystallographic R-factor of just under 19%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side-chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no significant internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by A. D. McLachlan [(1979) J. Mol. Biol. 133, 557-563] for soybean trypsin inhibitor.     

Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex (2:1) at 2.3 a resolution

The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 A resolution to a final R-factor of 0.177. A distance of 37.2 A between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten beta-strands and the loops connecting these strands but it lacks alpha-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel beta-strands, beta 1/beta 2 and beta 6/beta 7. Strands beta 1 and beta 6 are in direct contact with trypsin molecules and form stable triple stranded beta-sheet structures via hydrogen bonds.     

Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation.

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 A resolution.

The three-dimensional structure of telokin, an acidic protein identical to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard, has been determined at 2.8 A resolution and refined to a crystallographic R-factor of 19.5% for all measured X-ray data from 30 A to 2.8 A. Crystals used in the investigation belonged to the space group P3(2)21, with one molecule per asymmetric unit and unit cell dimensions of a = b = 64.4 A and c = 50.6 A. Telokin contains 154 amino acid residues, 103 of which were visible in the electron density map. The overall molecular fold of telokin consists of seven strands of antiparallel beta-pleated sheet that wrap around to form a barrel. There is also an extended tail of eight amino acid residues at the N terminus that does not participate in beta-sheet formation. The beta-barrel can be simply envisioned as two layers of beta-sheet, nearly parallel to one another, with one layer containing four and the other three beta-strands. This type of beta-barrel, as seen in telokin, was first observed for the CH2 domain of an immunoglobulin fragment Fc. Telokin is an intracellular protein and, as such, does not contain the disulphide linkage between beta-strands B and F normally observed in the immunoglobulin constant domains. It does, however, contain two cysteine amino acid residues (Cys63 and Cys115) that are situated at structurally identical positions to those forming the disulphide linkage in the immunoglobulin constant domain.     

Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors

Canonical serine protease inhibitors interact with cognate enzymes through the P3-P2' region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETI(L)-WCI(S), having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETI(L)-WCI(S)), where the barrier is reintroduced on ETI(L)-WCI(S), shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETI(L)-WCI(S)) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.     

Functional implications of interleukin-1 beta based on the three-dimensional structure.

The molecular structure of interleukin-1 beta, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. The framework of this molecule consists of 12 antiparallel beta-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a beta-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the beta-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.     

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed “mini‐proteins,” containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop‐scaffold compatibility, we prepared three chimeric proteins ECI^L‐WCI^S, ETI^L‐WCI^S, and STI^L‐WCI^S, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI^L‐WCI^S and STI^L‐WCI^S behave like good inhibitors, ETI^L‐WCI^S behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI^L‐WCI^S shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI^L‐WCI^S leading to a loss of canonical conformation, explaining its substrate‐like behavior. Incorporation of this barrier back in ETI^L‐WCI^S through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop‐scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.     

Recognition and stabilization of a unique CPRI--structural motif in cucurbitaceae family trypsin inhibitor peptides: molecular dynamics based homology modeling using the X-ray structure of MCTI-II

The high resolution crystallographic structure of MCTI-II complexed with beta trypsin (PDB entry 1MCT) was used to model the corresponding structures of the six inhibitor peptides belonging to Cucurbitaceae family (MCTI-I, LA-1, LA-2, CMTI-I, CMTI-III, CMTI-IV). Two model inhibitors, LA-1 and LA-2 were refined by molecular dynamics to estimate the average solution structure. The difference accessible surface area (DASA) study of the inhibitors with and without trypsin revealed the Arginine and other residues of the inhibitors which bind to trypsin. The hydration dynamics study of LA1 and LA2 also confirm the suitability of water molecules at the active Arg site. Moreover, the presence of a unique 3D-structural motif comprises with the four CPRI residues from the amino terminal is thought to be conserved in all the six studied inhibitors, which seems essential for the directional fixation for proper complexation of the Arg (5) residue towards the trypsin S1-binding pocket. The role of the disulphide linkage in the geometrical stabilization of CPRI (Cysteine, Proline, Arginine, Isoleucine) motif has also been envisaged from the comparative higher intra molecular Cys (3) -Cys (20) disulphide dihedral energies.     

Crystal structure of yeast Cu,Zn superoxide dismutase. Crystallographic refinement at 2.5 A resolution.

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by 1H-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 A and 0.66 A, respectively. The structures consist of two short antiparallel beta-strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta-strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides.     

Crystal structure of a 16 kDa double-headed Bowman-Birk trypsin inhibitor from barley seeds at 1.9 A resolution.

The Bowman-Birk trypsin inhibitor from barley seeds (BBBI) consists of 125 amino acid residues with two inhibitory loops. Its crystal structure in the free state has been determined by the multiwavelength anomalous diffraction (MAD) method and has been refined to a crystallographic R-value of 19.1 % for 8.0-1.9 A data. This is the first report on the structure of a 16 kDa double-headed Bowman-Birk inhibitor (BBI) from monocotyledonous plants and provides the highest resolution picture of a BBI to date. The BBBI structure consists of 11 beta-strands and the loops connecting these beta-strands but it lacks alpha-helices. BBBI folds into two compact domains of similar tertiary structure. Each domain shares the same overall fold with 8 kDa dicotyledonous BBIs. The five disulfide bridges in each domain are a subset of the seven disulfide bridges in 8 kDa dicotyledonous BBIs. Two buried water molecules form hydrogen bonds to backbone atoms in the core of each domain. One interesting feature of this two-domain inhibitor structure is that the two P1 residues (Arg17 and Arg76) are approximately 40 A apart, allowing the two reactive-site loops to bind to and to inhibit two trypsin molecules simultaneously and independently. The conformations of the reactive-site loops of BBBI are highly similar to those of other substrate-like inhibitors. This structure provides the framework for modeling of the 1:2 complex between BBBI and trypsin.     

Crystal structure of a sweet tasting protein thaumatin I, at 1.65 A resolution.

The crystal structure of thaumatin I, a potently sweet protein isolated from the fruits of the West African shrub, Thaumatococcus danielli Benth, has been refined at a resolution better than 1.65 A using a combination of energy minimization and stereochemically restrained least-squares methods. The final model consists of all 207 amino acids, 28 alternate amino acid conformers and 236 waters, with a crystallographic R-factor of 0.145 for 19,877 reflections having F > 4 sigma F between 10.0 A and 1.65 A (R = 0.167 for all 24,022 reflections). The model has good stereochemistry, with root-mean-square deviations from ideal values for bond and angle distances of 0.014 A and 0.029 A, respectively. The estimated root-mean-square co-ordinate error is 0.15 A. The current model confirms the previously reported 3.1 A C alpha trace in both main chain connectivity and disulfide topology, including two disulfide bonds, that differed from the earlier reported biochemical determination. The structure contains three domains. The core of the molecule consists of an eleven-stranded, flattened beta-sandwich folded into two Greek key motifs. All beta-strands in this sandwich are antiparallel except the parallel N-terminal and the C-terminal strands. The average hydrogen bond length in this sandwich is 2.89 A, with an angle of 155.1 degrees. Two beta-bulges are found in one of the sheets. The second domain consists of two beta-strands forming a beta-ribbon and connected by an omega-loop, and contains a proline residue in cis conformation. This structural motif folds back against the main sandwich to form a smaller sandwich-like structure. The third domain is a disulfide-rich region stretching away from the sandwich portion of the molecule. It contains one alpha-helix and three short helical fragments. Two of the helical segments are connected by an unusually sharp turn, stabilized by a disulfide bridge. One of the three disulfide bonds in this domain takes on two conformations.     

A general model of the P2 protein of peripheral nervous system myelin based on secondary structure predictions, tertiary folding principles, and experimental observations

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.     

Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate

Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion. I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands. The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics. The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees. I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets. The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation. The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules. The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16. The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues. The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.     

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.     

The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin.

Orthorhombic crystals of the complex formed between bovine alpha-chymotrypsin and a recombinant human mucous proteinase inhibitor (SLPI) were grown. Data to 2.3 A resolution were collected on the area-detector diffractometer FAST. The crystal structure of the complex was solved by Patterson search techniques using chymotrypsin as a search model. A cyclic procedure of modeling and crystallographic refinement enabled the determination of the SLPI structure. The current crystallographic R-value is 0.19. SLPI has a boomerang-like shape with both wings comprising two well separated domains of similar architecture. In each domain the polypeptide chain is arranged like a stretched spiral. Two internal strands form a regular beta-hairpin loop which is accompanied by two external strands linked by the proteinase binding segment. The polypeptide segment of each domain is interconnected by four disulfide bridges with a connectivity pattern hitherto unobserved. The reactive site loop of the second domain has elastase and chymotrypsin binding properties. It contains the scissile peptide bond between Leu72I and Met73I and has a similar conformation to that observed in other serine proteinase protein inhibitors. Eight residues of this loop, two of the adjacent hairpin loop, the C-terminal segment and Trp30I are in direct contact with the cognate enzyme. The binding loop of the first domain (probably with anti-trypsin activity) is disordered due to proteolytic cleavage occurring in the course of crystallization.     

Enzymatic cyclization of a potent bowman-birk protease inhibitor,sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.