Crystal Structure of Mung Bean Inhibitor Lysine Active Fragment Complex with Bovine β-trypsin at 1.8Å Resolution

Abstract

The crystal structure of the complex of mung bean inhibitor lysine active fragment with bovine β-trypsin has been determined by X-ray crystallographic analysis at a resolution of 1.8 Å. Refinement of the model of the complex converged at a final R value of 0.16. From the resulting electron density map, about one-third of the residues of the inhibitor were identified and two residues, at position P4 and P2′ respectively, were found to be inconsistent with the sequence reported previously. The peptide chain of the inhibitor at the trypsin active site turns back sharply at Pro23I and forms a 9-residue reactive loop, which interacts with trypsin in a similar manner to the other families of inhibitors, suggesting an important and common role of these regions in exhibiting inhibitory activity.     

Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz).

All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.     

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman–Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P4_1, with R_work/R_free values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme–inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.     

Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin

The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.     

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 at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).     

Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 Å resolution: Structure solution,crystallographic refinement and preliminary structural interpretation

The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex (Papamokos et al., 1982). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981). The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex (Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.     

Catalytic domain structures of MT-SP1/matriptase, a matrix-degrading transmembrane serine proteinase.

The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 A and bovine pancreatic trypsin inhibitor at 2.9 A. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.     

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Tyrosine Sulfation of Human Trypsin Steers S2’ Subsite Selectivity towards Basic Amino Acids

Human cationic and anionic trypsins are sulfated on Tyr154, a residue which helps to shape the prime side substrate-binding subsites. Here, we used phage display technology to assess the significance of tyrosine sulfation for the specificity of human trypsins. The prime side residues P1′–P4′ in the binding loop of bovine pancreatic trypsin inhibitor (BPTI) were fully randomized and tight binding inhibitor phages were selected against non-sulfated and sulfated human cationic trypsin. The selection pattern for the two targets differed mostly at the P2′ position, where variants selected against non-sulfated trypsin contained primarily aliphatic residues (Leu, Ile, Met), while variants selected against sulfated trypsin were enriched also for Arg. BPTI variants carrying Arg, Lys, Ile, Leu or Ala at the P2′ position of the binding loop were purified and equilibrium dissociation constants were determined against non-sulfated and sulfated cationic and anionic human trypsins. BPTI variants harboring apolar residues at P2′ exhibited 3–12-fold lower affinity to sulfated trypsin relative to the non-sulfated enzyme, whereas BPTI variants containing basic residues at P2′ had comparable affinity to both trypsin forms. Taken together, the observations demonstrate that the tyrosyl sulfate in human trypsins interacts with the P2′ position of the substrate-like inhibitor and this modification increases P2′ selectivity towards basic side chains.     

Three-dimensional structure of the complexes between bovine chymotrypsinogen A and two recombinant variants of human pancreatic secretory trypsin inhibitor (Kazal-type)

Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both crystallize isomorphously in space group P4(1)2(1)2 with lattice constants a = 84.4 A, c = 86.7 A and diffract to 2.3 A resolution. The structure was solved by molecular replacement. The final R-value after refinement with 8.0 to 2.3 A resolution data was 19.5% for both complexes after inclusion of about 50 bound water molecules. The overall three-dimensional structure of PSTI is similar to the structure of porcine PSTI in the trypsinogen complex (1TGS). Small differences in the relative orientation of the binding loop and the core of the inhibitors indicate flexible adaptation to the proteases. The chymotrypsinogen part of the complex is similar to chymotrypsin. After refolding induced by binding of the inhibitor the root-mean-square difference of the active site residues A186 to A195 and A217 to A222 compared to chymotrypsin was 0.26 A.     

Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition

We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.     

Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution. Structural basis of Janus-faced serine protease inhibitor specificity

Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance. In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity. The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin. In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement. Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region. Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin. However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment. This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents.     

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.     

Potato I型蛋白酶抑制剂 rBTI及其突变体的表达、纯化和活性研究

荞麦胰蛋白酶抑制剂(BTI)属于丝氨酸蛋白酶抑制剂Potato I家族,典型构象中有1段暴露在分子外侧的结合区,该区内P1′、P2、P6′与P8′位的氨基酸残基具有高度保守性.本文依据近期解析的rBTI晶体结构以及rBTI与胰蛋白酶复合物晶体结构信息,对rBTI 中的P2和P8′位氨基酸进行突变,构建了pExSecI- Bti-P44T 和 pExSecI-Bti-W53R 重组质粒,转入大肠杆菌 BL21（DE3）中进行表达,通过Resource Q阴离子交换层析和Superdex G 75 HR 10/300凝胶柱进行分离纯化后,测定了rBTI及其突变体对胰蛋白酶的抑制常数,以及它们对HepG2 细胞内的蛋白酶体和细胞增殖的抑制作用. 实验结果显示,rBTI 的44和53位分别突变为 Thr和Arg后,Ki 分别为2.91×10-9mol/L和2.97 ×10-7mol /L,前者较rBTI（Ki = 3.56×10-8 mol/L）降低1个数量级,而后者较rBTI升高1个数量 级.功能分析显示,rBTI及2种突变体对HepG2细胞内的蛋白酶体基本没有抑制作 用,但是它们都保留了对HepG2细胞增殖的抑制活性.这些结果揭示,作为一种特异的胰蛋白酶抑制剂,rBTI分子中的保守区域氨基酸残基虽然对胰蛋白酶的抑制作用有显著影响,但并不影响其抑制肿瘤细胞的增殖,仍能发挥其原有的生物学功能.     

Coevolution of insect trypsins and inhibitors

Many plant proteinase inhibitors have lysine at the P1 position, presumably to avoid hydrolysis by insect trypsins. Lepidopteran trypsins appear to have adapted to resist proteinase inhibitors through increased inhibitor hydrolysis and decreased binding to inhibitor hydrophilic reactive sites. Lepidopteran digestive trypsins prefer lysine at the P1 position and have substrate binding subsites more hydrophobic than trypsins from insects in other orders. All available sequences of sensitive and inhibitor-insensitive insect trypsins were aligned with porcine trypsin, for which interactions with Kunitz and Bowman-Birk inhibitor are known. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the following residues were considered important to insect trypsin-PI interactions (chymotrypsin numbering): 60, 94, 97, 98, 99, 188, 190, 213, 215, 217, 219, 228. These residues support the Neighbor Joining analysis tree branches separating sensitive and insensitive trypsin sequences. Primary sequences interacting with PIs are around the active site, with some forming part of the S1 (188, 217, 219 and 228) or S4 (99, 215) pockets.     

Selective inhibition of trypsins by insect peptides: role of P6-P10 loop

PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K(i) around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K(i) 相似文献   

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.