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Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors., (© 2024 The Protein Society.)

Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50., (© 2021 The Protein Society.)

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM  = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases., (© 2015 The Protein Society.)

Covalently linked pairs of well-chosen peptides can be good model systems for protein folding studies because they can adopt stable secondary, side-chain, and tertiary structure under certain conditions. We demonstrate a method for characterizing the structure in such peptide pairs by hydrogen/deuterium exchange of individual amide groups analyzed by collision-induced dissociation tandem mass spectrometry, in concert with circular dichroism spectroscopy. We apply the method to two peptides (and their three possible pairs) from bovine pancreatic trypsin inhibitor to address specific hypotheses regarding the stabilization of local secondary structure by long-range interactions.

The binding of P1 variants of bovine pancreatic trypsin inhibitor (BPTI) to trypsin has been investigated by means of molecular dynamics simulations. The specific interaction formed between the amino acid at the primary binding (P1) position of the binding loop of BPTI and the specificity pocket of trypsin was estimated by use of the linear interaction energy (LIE) method. Calculations for 13 of the naturally occurring amino acids at the P1 position were carried out, and the results obtained were found to correlate well with the experimental binding free energies. The LIE calculations rank the majority of the 13 variants correctly according to the experimental association energies and the mean error between calculated and experimental binding free energies is only 0.38 kcal/mole, excluding the Glu and Asp variants, which are associated with some uncertainties regarding protonation and the possible presence of counter-ions. The three-dimensional structures of the complex with three of the P1 variants (Asn, Tyr, and Ser) included in this study have not at present been solved by any experimental techniques and, therefore, were modeled on the basis of experimental data from P1 variants of similar size. Average structures were calculated from the MD simulations, from which specific interactions explaining the broad variation in association energies were identified. The present study also shows that explicit treatment of the complex water-mediated hydrogen bonding network at the protein-protein interface is of crucial importance for obtaining reliable binding free energies. The successful reproduction of relative binding energies shows that this type of methodology can be very useful as an aid in rational design and redesign of biologically active macromolecules.

We develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded "decoys." This is due in part to the fact that the Coulomb energy of the native protein is consistently lower than that of the decoys. The solvation free energy generally favors the decoys, although the total electrostatic free energy (sum of Coulomb and solvation terms) favors the native structure. The behavior of the solvation free energy is somewhat counterintuitive and, surprisingly, is not correlated with differences in the burial of polar area between native structures and decoys. Rather. the effect is due to a more favorable charge distribution in the native protein, which, as is discussed, will tend to decrease its interaction with the solvent. Our results thus suggest, in keeping with a number of recent studies, that electrostatic interactions may play an important role in determining the native topology of a folded protein. On this basis, a simplified scoring function is derived that combines a Coulomb term with a hydrophobic contact term. This function performs as well as the more complete free energy evaluation in distinguishing the native structure from misfolded decoys. Its computational efficiency suggests that it can be used in protein structure prediction applications, and that it provides a physically well-defined alternative to statistically derived scoring functions.

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.

The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174-Arg 175 by Gly 174-Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D-Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A-Thr 60I insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D-Phe and v-Tyr residues.

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.

The 3-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor Ecballium elaterium (EETI-II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE-derived distance constraints. Spectra were assigned using 2-dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three-bond spin-spin couplings between C alpha H and amide protons, amide exchange rates, and the temperature dependence of amide chemical shifts were also measured. The structure consists largely of loops and turns, with a short region of beta-sheet. The Leu-5 substitution produces a substantial reduction in affinity for trypsin relative to native EETI-II, which contains an Ile at this position. The global structure of the Leu-5 analogue studied here is similar to that reported for native EETI-II (Heitz A, Chiche L, Le-Nguyen D, Castro B, 1989, Biochemistry 28:2392-2398) and to X-ray and NMR structures of the related proteinase inhibitor CMTI-I (Bode W et al., 1989, FEBS Lett 242:285-292; Holak TA et al., 1989a, J Mol Biol 210:649-654; Holak TA, Gondol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635-648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991, J Am Chem Soc 113:3196-3198). The region near the scissile bond is the most disordered part of the structure, based on geometric superimposition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest of the protein. This motional disorder is increased in the Leu-5 analogue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (D) amino acids was also studied by NMR and found to have a spectrum identical with that of the (L) form. This is consistent with the (D) form being a mirror image of the (L) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (D) form has no activity against trypsin, as would be expected for a mirror-image form.

Crystal structures of four mutants of bovine pancreatic trypsin inhibitor (F22A, Y23A, N43G, and F45A), engineered to alter their stability properties, have been determined. The mutated residues, which are highly conserved among Kunitz-type inhibitors, are located in the rigid core of the molecule. Replacement of the partially buried bulky residues of the wild-type protein with smaller residues resulted in crevices open to the exterior of the molecule. The overall three-dimensional structure of these mutants is very similar to that of the wild-type protein and only small rearrangements are observed among the atoms lining the crevices.

The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment.