Protease-binding Loop Research Articles

Canonical or noncanonical? Structural plasticity of serine protease-binding loops in Kunitz-STI protease inhibitors.

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the β-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4-β5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the β5-β6 and β9-β10 loops. In addition, we found that the β5-β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

Scaffold stability and P14' residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4.

Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14′ position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.

A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis.

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin

Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

Inhibition of Kallikrein-Related Peptidases 7 and 5 by Grafting Serpin Reactive-Center Loop Sequences onto Sunflower Trypsin Inhibitor-1 (SFTI-1).

Serpin proteins irreversibly inhibit serine proteases, but only a small part of the serpin reactive-center loop (RCL) is responsible for the initial protein-protein interaction (PPI). To develop peptidic protease inhibitors, kallikrein-related peptidases 7 (KLK7) and 5 (KLK5) were chosen. Firstly, we demonstrated that short peptides derived from RCL sequences can be cleaved by KLK7 in a substrate-like manner. Next, these substrates were grafted onto the protease-binding loop of sunflower trypsin inhibitor-1 (SFTI-1). Peptides based on kallistatin, α1 -antichymotrypsin, and protein C inhibitor (PCI) inhibited KLK7 with Ki =0.4, 0.5, and 0.7 μm, respectively. In contrast, the trypsin-like KLK5 was only blocked by the peptide derived from PCI (Ki =0.6 μm). Thus, serpin function can be mimicked by introducing its PPI site into the rigid structure of the SFTI-1 scaffold. This approach might be applicable not only to KLKs but also to other serine protease members, thus opening up new therapeutic fields.

New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its K i for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

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 Ki 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.

Interactions Outside the Proteinase-binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by Its Canonical Inhibitor from Corn

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.

Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

Reconciling the lock-and-key and dynamic views of canonical serine protease inhibitor action

The efficiency of canonical serine protease inhibitors is conventionally attributed to the rigidity of their protease binding loop with no conformational change upon enzyme binding, yielding an example of the lock-and-key model for biomolecular interactions. However, solution-state structural studies revealed considerable flexibility in their protease binding loop. We resolve this apparent contradiction by showing that enzyme binding of small, 35-residue inhibitors is actually a dynamic conformer selection process on the nanosecond-timescale. Thus, fast timescale dynamics enables the association rate to be solely diffusion-controlled just like in the rigid-body model.

An Ion-channel Modulator from the Saliva of the Brown Ear Tick has a Highly Modified Kunitz/BPTI Structure

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 Å resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by β-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the ”Kunitz head” by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a β-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.

Local binding with globally distributed changes in a small protease inhibitor upon enzyme binding

Complexation of the small serine protease inhibitor Schistocerca gregaria chymotrypsin inhibitor (SGCI), a member of the pacifastin inhibitor family, with bovine chymotrypsin was followed by NMR spectroscopy. (1)H-(15)N correlation (HSQC) spectra of the inhibitor with increasing amounts of the enzyme reveal tight and specific binding in agreement with biochemical data. Unexpectedly, and unparalleled among canonical serine protease inhibitors, not only residues in the protease-binding loop of the inhibitor, but also some segments of it located spatially far from the substrate-binding cleft of the enzyme were affected by complexation. However, besides changes, some of the dynamical features of the free inhibitor are retained in the complex. Comparison of the free and complexed inhibitor structures revealed that most, but not all, of the observed chemical shift changes can be attributed to minor structural transitions. We suggest that the classical 'scaffold + binding loop' model of canonical inhibitors might not be fully valid for the inhibitor family studied. In our view, this feature allows for the emergence of both taxon-specific and nontaxon-specific inhibitors in this group of small proteins.

Structure-Function Relationships in Serine Protease-Bovine Pancreatic Trypsin Inhibitor Interaction

We report our progress in understanding the structure-function relationships for the interaction between BPTI and serine proteases. We focused on extensive mutagenesis of four crucial positions from the protease binding loop of BPTI. Selected variants were characterized by determination of association constants, stability parameters and structures of protease-inhibitor complexes.

Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor

The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K(i) values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca(2+) at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca(2+) had weaker affinity for AMY2 and reduced sensitivity to the Ca(2+) modulation of the affinity. These results suggest that the Ca(2+) and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases.

Selection of potent chymotrypsin and elastase inhibitors from M13 phage library of basic pancreatic trypsin inhibitor (BPTI)

The combinatorial approach offered by phage display has proved to be powerful in obtaining novel variants of canonical inhibitors of serine proteinases that show new binding patterns. We applied this strategy to search for variants of basic pancreatic trypsin inhibitor (BPTI) that would be strong inhibitors of two serine proteinases: bovine α-chymotrypsin and porcine pancreatic elastase. BPTI only moderately inhibits the first and does not inhibit the second enzyme. A representative library of 3.2×10 4 BPTI variants, randomized at P 1, P 1′, P 2′ and P 3′ positions of the proteinase binding loop, was displayed on the surface of phage M13. After four to five rounds of selection on the target proteinase consensus sequences of the inhibitor binding loop were obtained. In both cases, the variants selected differed from BPTI at two to four positions, with a strong preference for selection of hydrophobic residues. Nevertheless, five of these variants expressed in a free form appeared to be correctly folded, stable proteins, and did not aggregate during thermal denaturation. The midpoints of the thermal unfolding curves of these variants were lowered by 5–20°C as compared to BPTI. The expressed variants proved to be new potent inhibitors of the target enzymes with association constants up to 6.9×10 9 M −1 and 3.7×10 10 M −1 for elastase and chymotrypsin, respectively. Thus, the inhibitory properties of BPTI were improved by as much as 7×10 6-fold towards elastase and 420-fold towards chymotrypsin.

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine alpha-chymotrypsin at 2.1- and 3.0-A resolution, respectively. PMP-C is a potent bovine alpha-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine alpha-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel beta-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P'4, corresponding to residues 26-34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of alpha-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P'4).

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks

The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of β-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3Jαβcoupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91 %) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 Å (backbone atoms) and 1.02 Å (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel β-sheet, a single α-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6′ and P8′, respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the Arg→Trp substitution at P8′ is a slightly more compact conformation of the loop relative to the protein core.

Design, Chemical Synthesis and Kinetic Studies of Trypsin Chromogenic Substrates Based on the Proteinase Binding Loop of Cucurbita maxima Trypsin Inhibitor (CMTI-III)

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P4 on the substrate–enzyme interaction, kinetic parameters of newly synthesized substrates with bovine β-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P4 significantly enhanced the substrate specificity (kcat/Km) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine β-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.