The structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor

The structure of the complex between anhydro-trypsin and pancreatic trypsin inhibitor has been determined by difference Fourier techniques using phases obtained from the native complex (Huber et al., 1974). It was refined independently by constrained crystallographic refinement at 1.9 å resolution. The anhydro-complex has Ser 195 converted to dehydro-alanine. There were no other significant structural changes. In particular, the high degree of pyramidalization of the C atom of Lys 15 (I) of the inhibitor component observed in the native complex is maintained in the anhydro-species.     

Reactive sites of an anticarcinogenic Bowman-Birk proteinase inhibitor are similar to other trypsin inhibitors.

The three-dimensional structure of the Bowman-Birk type proteinase inhibitor (PI-II) has been determined by x-ray crystallography and refined at 2.5-A resolution. This protein is a specific inhibitor of trypsin. Two reactive site loops, one at each end of the PI-II molecule, are structurally similar to each other and to reactive-site loops of pancreatic secretory trypsin inhibitor (Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1982) J. Mol. Biol. 162, 839-869) and bovine pancreatic trypsin inhibitor (Deisenhofer, J., and Steigemann, W. (1975) Acta Crystallogr. B31, 238-250). PI-II is the first reported Bowman-Birk type inhibitor structure to be refined at high resolution, providing further insight into inhibitor mechanisms.     

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution

The complex formed by bovine trypsinogen and the pancreatic trypsin inhibitor crystallizes in large crystals isomorphous with trypsin-PTI² complex crystals Rühlmann et al. 1973. X-ray diffraction data to 1.9 Å resolution were collected in the absence and presence of Ile-Val dipeptide. Both trypsinogen complex structures have been crystallographically refined, using the refined trypsin-PTI complex Huber et al. 1974a as a starting model. The final R values are 0.25 and 0.26, respectively. The mean main-chain atom deviations between the three complex structures are about 0.15 Å. In contrast, the mean deviation between the complexed and the free trypsinogen Fehlhammer et al. 1977 is 0.28 Å, reflecting the influence of crystal packing and complexation. The trypsinogen component adopts a trypsin-like conformation upon PTI binding: The Asp194 side-chain turns around and the activation domain becomes rigid, forming the specificity pocket and the Ile16 binding cleft. The specific interactions between PTI and trypsin are also observed in the trypsinogen complex. As in free trypsinogen, the N-terminus including residues Val10 to Gly18 is mobile and sticks out into solution. Apart from the different arrangement of the N-termini in the two complexes, the only significant, but minor structural difference is the enhanced thermal mobility of the autolysis loop in the trypsinogen complex. Upon binding of the Ile-Val dipeptide, the autolysis loop becomes fixed as in the trypsin complex. The Ile-Val position is identical in the ternary and the trypsin complex.     

The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

The structure of the variable portions of a K-type Bence-Jones protein REI forming a dimer has been determined by X-ray diffraction to a resolution of 2.0 A. The structure has been refined using a constrained crystallographic refinement procedure. The final R value is 0.24 for 15000 significantly measured reflections; the estimated standard deviation of atomic positions is 0.09 A. A more objective assessment of the error in the atomic positions is possible by comparing the two independently refined monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45, 513). Now a detailed analysis is presented in terms of hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta structure, reverse turns) are defined. A more precise atomic arrangement of the amino acid residues forming the contact region and the hapten binding site is given as well as the localization of solvent molecules. Two cis-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular structure enables crystal structure analyses of related molecules to be made by Patterson search techniques. The calculated phases based on the refined structure are much improved compared to isomorphous phases. Therefore the effects of hapten binding on the molecular structure can be analyzed by the difference Fourier technique with more reliability. Hapten binding studies have been started.     

Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4₁2₁2 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined.A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen &; Bode. 1983).The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen &; Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

Comparison of two highly refined structures of bovine pancreatic trypsin inhibitor

The high resolution structures of bovine pancreatic trypsin inhibitor refined in two distinct crystal forms have been compared. One of the structures was a result of new least-squares X-ray refinement of data from crystal form I, while the other was the joint X-ray/neutron structure of crystal form II. After superposition, the molecules show an overall root-mean-squares deviation of 0.40 A for the atoms in the main chain, while the deviations for the side-chain atoms are 1.53 A. The latter number decreases to 0.61 A when those side-chains that adopted drastically different conformations are excluded from comparison. The discrepancy between atomic temperature factors in the two models was 6.7 A2, while their general trends are highly correlated. About half of the solvent molecules occupy similar positions in the two models, while the others are different. As expected, solvents with the lowest temperature factors are most likely to be common in the two crystal forms. While the two models are clearly similar, the differences are significantly larger than the errors inherent in the structure determination.     

Crystal structure of the human Fab fragment Kol and its comparison with the intact Kol molecule

The crystal structure of the Fab³ fragment of the IgG protein Kol was analysed and an electron density map calculated at 3 Å resolution based on phases obtained from multiple isomorphous replacement. An atomic model was constructed but the lack of amino acid sequence data causes some uncertainty, in particular concerning the amino acid side-chains. Several cycles of constrained crystallographic refinement (Deisenhofer &; Steigemann, 1975) produced an R value of 0.36. The resulting model was compared with the intact Kol IgG crystal structure (Colman et al., 1976), which had also been subjected to constrained crystallographic refinement, and two Fab fragments (Davies et al., 1975). The elbow angle was found open and only 8 ° more bent than in intact Kol, in contrast to the other Fab fragments, which show a strongly bent elbow angle. The intramolecular longitudinal V-C contacts are very similar in Kol Fab and Kol IgG and considerably fewer in number than in the other Fab fragments. The lateral V-V and C-C association is virtually identical in Kol Fab and Kol IgG. Although Kol Fab and Kol IgG crystallize in different lattices, they exhibit a nearly identical head to tail packing of the Fab parts, with the C modules touching the hypervariable segments of the V modules. This strongly conserved particular mode of aggregation might be reflected in the property of cold precipitation of the Kol cryoglobulin.     

Crystallographic refinement of Japanese quail ovomucoid, a Kazal-type inhibitor, and model building studies of complexes with serine proteases

Japanese quail ovomucoid third domain (OMJPQ3), a Kazal-type inhibitor, was crystallographically refined with energy constraints. The final R-value is 0.20 at 1.9 Å resolution. The four molecules in the asymmetric unit are very similar, with deviations of main-chain atoms between 0.2 and 0.3 Å. An analysis of the side-chain hydrogen-bonding pattern and amino acid variability in the Kazal family shows a high correlation between hydrogen-bonding and conservation.The conformation of the reactive site loop (P2-P2′) of OMJPQ3 is similar to those of basic pancreatic trypsin inhibitor, Streptomyces subtilisin inhibitor, and soybean trypsin inhibitor. This suggests a common binding mode and justifies model-building studies of complexes.Complexes of OMJPQ3 with trypsin, chymotrypsin and elastase were modelled on the basis of the trypsin-basic pancreatic trypsin inhibitor complex structure and inspected by use of a computer graphics system. Stereochemically satisfying models were constructed in each case and detailed interactions are proposed. The complex with elastase is of particular interest, showing that leucine and methionine are good P1 residues. A good correlation is observed between functional properties of ovomucoid variants and the position of the exchanged residues with respect to the modelled inhibitor-protease contact.     

Crystal structure of alpha-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor.

The three-dimensional structure of alpha-dendrotoxin (alpha-DTX) from the green mamba (Dendroaspis angusticeps) venom has been determined crystallographically using the method of isomorphous replacement and refined at 2.2 A resolution using a restrained least-squares method. The crystallographic R-factor is 0.169 for all 3451 measured reflections between 7.0 and 2.2 A. Although the main-chain fold of alpha-DTX is similar to that of homologous bovine pancreatic trypsin inhibitor (BPTI), there are significant differences involving segments of the polypeptide chain close to the "antiprotease site" of BPTI. Comparison of the structure of alpha-DTX with the existing models of BPTI and its complexes with trypsin and kallikrein reveals structural differences that explain the inability of alpha-DTX to inhibit trypsin and chymotrypsin.     

Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor.

The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R-factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

Flexibility of bovine pancreatic trypsin inhibitor

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.     

1．9A分辨率Al－（L－丙氨酸）胰岛素晶体结构研究

使用Ｘ－ＰＬＯＲ及立体化学制约最小二乘精化技术,并结合差值Ｆｏｕｒｉｅｒ图人工分析,测定了１．９　分辨率Ａｌ修饰丙氨酸胰岛素的晶体结构,晶体空间群为Ｒ３,晶胞参数：ａ＝ｂ＝８０．８９,ｃ＝３７．６４。精化后的结构模型最终偏离因子Ｒ＝０．１８５,同理想键长和键角的均方根偏差分别为０．０１８和３．５°,独立区内二个分子的Ａ链Ｎ端Ａｌ－丙氨酸残基清晰可见。     

Complex formation by bovine trypsin and a tetrapeptide (Leu-Arg-Pro-Gly-NH2): X-ray structure analysis of the complex in the orthorhombic crystal form with low molecular packing density

The title tetrapeptide, Leu-Arg-Pro-Gly-NH₂, forms a complex with trypsin in a novel orthorhombic crystal form with low molecular packing density. The complex formation was directly evidenced by X-ray crystallography. The crystal structure at 1.8 Å resolution was refined to anR-factor of 20.5% for 13,923 reflection data, which were measured with synchrotron radiation. The tetrapeptide is bound to trypsin at the active site, and the binding mode is very similar to that of a bovine pancreatic trypsin inhibitor (BPTI):trypsin complex. The tetrapeptide:trypsin complex is the first observation that a peptide forms a stable complex with trypsin.     

Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution.

The crystal structure of the bacterial serine protease from Streptomyces griseus (SGPA) has been refined at 1.8 Å resolution by a restrained parameter least-squares procedure (Konnert, 1976) to a conventional R factor of 0.139 for 12662 statistically significant reflections [I > 3σ(I)]. The number of variable parameters in the final model was 5912 which included positional and individual thermal parameters of the enzyme, and positions, B factors and occupancies of 175 solvent molecules. The algorithm used for this refinement allows for the simultaneous restraint on bond distances and distances related to interbond angles, the coplanarity of atoms in planar groups, the conservation of chirality of asymmetric centres, non-bonded contact distances, conformational torsional angles and individual isotropic temperature factors.The refined structure of SGPA differs from ideal bond lengths by an overall root-mean-square deviation of 0.02 Å; the corresponding value for angle distances is 0.038 Å. Comparison of the phase angles for the shell of data, 8.0 to 2.8 Å, between the multiple isomorphous replacement phases (Brayer et al., 1978a) and the refined phases, indicates an overall difference (r.m.s.) of 56.6 °. The average conformational angle of the peptide bond (ω) is 179.7 ° (root-mean-square deviation ± 2.5 °) for the 180 peptide bonds of SGPA. Of the 175 solvent molecules included during the course of the refinement, 22 with occupancies ranging from 1.00 to 0.38 are located in the active site and the substrate binding region. It was not until these water molecules were included in the refinement process that the active Ser195 adopted its final conformation (χ¹ = ?77 °). The resulting distance from O^γ of Ser195 to N^ε2 of His57 is 3.1 Å, which, when taken with the observed distortion from linearity (50 °), indicates a rather weak interaction.     

The atomic structure of crystalline porcine pancreatic elastase at 2.5 A resolution: comparisons with the structure of alpha-chymotrypsin

Three isomorphous heavy-atom derivatives have been used to calculate a 2.5 Å resolution electron density map of tosyl-elastase at pH 5.0, from which an accurate atomic model has been constructed. Atomic co-ordinates measured from this model have been refined using model building, real-space refinement and energy minimization programs. The three-dimensional conformation of the polypeptide chain is described in terms of conformational angles, hydrogen-bonding networks and the environment of different types of amino acid side-chain.Difference Fourier calculation of the high resolution structure of native elastase at pH 5.0 shows it to be virtually identical to that of the tosyl derivative, except near the tosyl group. The conformation of the catalytically important residues in native elastase is very similar to that of native α-chymotrypsin, except for the orientation of the active centre serine oxygen. The significance of important structural similarities and differences between these two enzymes is discussed.Elastase contains 25 internal water molecules which play an important role in stabilizing the active conformation of the enzyme. Many of these water molecules are in identical positions to those found in the interior of α-chymotrypsin     

Atomic resolution crystal structure of HV‐BBI protease inhibitor from amphibian skin in complex with bovine trypsin

Protease inhibitors of the Bowman‐Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV‐BBI) in complex with trypsin. HV‐BBI shares significant similarities in sequence with a previously described inhibitor from a diskless‐fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher K_i against trypsin than HV‐BBI. Comparative analysis of trypsin cocrystal structures of HV‐BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI‐1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease‐binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin‐like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc.     

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.