Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.     

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d ‐Phe13Ala, and l ‐Phe13 variants. Structure‐activity relationship analysis indicated that d ‐Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

It has recently been shown that the biological activity of the second generation kinin B1 receptor selective antagonist, desArg10 HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg10 HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide DTic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg10 HOE 140 to that of an agonist.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. I. Conformations of the residue and of dipeptides

The L-azetidine-2-carboxylic acid (Aze) residue can be incorporated into proteins in the place of L-proline, of which it is the lower homologue. This substitution alters the properties of proteins, especially of collagen. Conformational constraints in N-acetyl-Aze-N'-methylamide and in several dipeptides containing Aze have been analyzed by means of energy computations. They have been compared with peptides containing Pro. The overall conformational preferences of Aze and Pro are similar, but several significant differences occur between them. In general, peptides containing Aze are somewhat more flexible than corresponding peptides containing Pro, because of a decrease in constraints caused by repulsive nonconvalent interactions of the atoms of the ring with neighboring residues. This results in an entropic effect that lessens the stability of ordered polypeptide conformations with respect to the disordered statistical coil. The collagen-like near-extended conformation is energetically less favorable for Aze than for Pro in the single residue and in dipeptides. This effect also contributes to a destabilization of the collagen triple helix. The influence of Aze on the conformation of polypeptides is discussed in the accompanying papers.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. IV. Local substitutions in the collagen triple helix

The properties of collagen are affected by the replacement of Pro by imino acid analogues. The structural effect of the low-level local substitution of L -azetidine-2-carboxylic acid (Aze) has been analyzed by computing the energy of CH₃CO-(Gly-Pro-Pro)₄-NHCH₃ triple helices in which a single residue of one strand has been replaced by Aze. When Aze is in position Y of a (Gly-X-Y) unit, low-energy local deformations are introduced in the triple helix, i.e., it becomes more flexible. On the other hand, the flexibility of the triple helix is not increased with Aze in position X. The energy of the triple helix to coil transition is not changed significantly by this amount of substitution. In an earlier study, we have demonstrated that the regular substitution of Aze in every tripeptide distorts or destabilizes the triple helix to a large extent [A. Zagari, G. Némethy, & H. A. Scheraga (1990) Biopolymers, Vol. 30, pp. 967–974 ]. Thus, it appears that a high level of substitution is required to cause the observed chemical and biological effects of Aze on collagen. © 1994 John Wiley & Sons, Inc.     

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by (1)H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 A, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H(alpha) protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.     

Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases

Fourteen monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds were synthesized by the solid-phase method. The purpose of this work was to establish the role of a disulfide bridge present in inhibitor’s side chains of Cys3 and Cys11 in association with serine proteinases. This cyclic fragment was replaced by the disulfide bridges formed by l-pencillamine (Pen), homo-l-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or combination of the three with Cys. As in the substrate specificity the P₁ position of the synthesized analogues Lys, Nlys [N-(4-aminobutyl)glycine], Phe or Nphe (N-benzylglycine) were present, and they were checked for trypsin and chymotrypsin inhibitory activity. The results clearly indicated that Pen and Nhcy were not acceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11) could be substituted without any significant impact on the affinity towards proteinase. On the other hand, elongation of the Cys3 side chain by introduction of Hcy did not affect inhibitory activity, and an analogue with the Hcy–Hcy disulfide bridge was more than twice as effective as the reference compound ([Phe⁵] SFTI-1) in inhibition of bovine α-chymotrypsin.     

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

Summary It has recently been shown that the biological activity of the second generation kinin B₁ receptor selective antagonist, desArg¹⁰ HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg¹⁰ HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide Dtic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg¹⁰ HOE 140 to that of an agonist.     

Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.     

The P1' specificity of tobacco etch virus protease

Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. II. Homopolymers and copolymers

The alteration of polymer conformational properties caused by the replacement of L-proline by L-azetidine-2-carboxylic acid (Aze) has been studied by means of conformational energy computations. In addition to poly(Aze), two sequential copolymers, poly(Pro-Aze) and poly(Aze3-Pro3), have been investigated. All polymers containing Aze are more flexible than poly(Pro). This is a consequence of an increased number of permitted conformational states for the Aze residue, as compared to Pro, when they are incorporated into a polypeptide, as well as of a lessened cooperativity of the trans-cis transition. The results of the computation can be used to interpret the observed physical properties of poly(Aze) and of its copolymers.     

The absolute structural requirement for a proline in the P3'-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.     

Specificity of prolyl endopeptidase

A series of tetrapeptides, Cbz(Bz)-Gly-X-Leu-Gly, were synthesized and the kinetic parameters, kcat and kcat/Km, determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N-Me-Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = alpha-aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, alpha-aminoisobutyryl, N-Me-Val, N-Me-Leu, Hyp(O-Bzl) and Ser(O-Bzl) were not cleaved at all. These results suggest that the specificity-determining site or S1 subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or C alpha which must not exceed the size of the pyrrolidine ring of proline.     

Peptomeric analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds

A series of linear and monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds, modified by N-(4-aminobutyl)glycine (Nlys) and N-benzylglycine (Nphe), were obtained by the solid-phase method. Some of these peptomers displayed trypsin or chymotrypsin inhibitory activity. In contradiction to the literature data, in most analogues peptide bonds formed by these peptoid monomers were at least partially hydrolyzed by the experimental enzymes at two different pH (3.5 and 8.3). Nevertheless, the replacement of Phe present in the P(1) substrate specificity of linear inactive SFTI-1 analogue with Nphe, yielded a potent chymotrypsin inhibitor. The introduction of one cyclic element (a disulfide bridge or head-to-tail cyclization) to the analogues synthesized significantly increased their proteinase resistance.     

Structure/function relationships in the inhibition of thimet oligopeptidase by carboxyphenylpropyl-peptides.

Some novel N-[1(RS)-carboxy-3-phenylpropyl]tripeptide p-aminobenzoates have been synthesised as inhibitors of thimet oligopeptidase (EC 3.4.24.15). These compounds are considered to bind as substrate analogues with the Cpp group in S1 and the peptide portion in the S' sites. The most potent inhibitor is Cpp-Ala-Pro-Phe-pAb, which has a Ki = 7 nM. Substitution of Gly for Ala at P1' leads to weaker binding which can be ascribed to increased rotational freedom. Good substrates often have Pro at P2' and Pro is favoured over Ala at this position in the inhibitors, too. When P2' is Pro, Phe is preferred over Tyr and Trp in P3'. The p-aminobenzoate group makes an important contribution to the binding, probably by forming a salt bridge, and removal of the C-terminal negative charge results in much less potent inhibitors.     

Insights into processing and cyclization events associated with biosynthesis of the cyclic Peptide kalata b1

Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1', and P2' positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1' position. The P2' position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2' Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.     

Interstrand dipole-dipole interactions can stabilize the collagen triple helix

The amino acid sequence of collagen is composed of GlyXaaYaa repeats. A prevailing paradigm maintains that stable collagen triple helices form when (2S)-proline (Pro) or Pro derivatives that prefer the C(γ)-endo ring pucker are in the Xaa position and Pro derivatives that prefer the C(γ)-exo ring pucker are in the Yaa position. Anomalously, an amino acid sequence in an invertebrate collagen has (2S,4R)-4-hydroxyproline (Hyp), a C(γ)-exo-puckered Pro derivative, in the Xaa position. In certain contexts, triple helices with Hyp in the Xaa position are now known to be hyperstable. Most intriguingly, the sequence (GlyHypHyp)(n) forms a more stable triple helix than does the sequence (GlyProHyp)(n). Competing theories exist for the physicochemical basis of the hyperstability of (GlyHypHyp)(n) triple helices. By synthesizing and analyzing triple helices with different C(γ)-exo-puckered proline derivatives in the Xaa and Yaa positions, we conclude that interstrand dipole-dipole interactions are the primary determinant of their additional stability. These findings provide a new framework for understanding collagen stability.