Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the...

Biochemical and Biophysical Research Communications 314 (2004) 755–765

BBRCwww.elsevier.com/locate/ybbrc

Molecular mechanism of enzyme inhibition: prediction of thethree-dimensional structure of the dimeric trypsin inhibitor from

Leucaena leucocephala by homology modelling

Rabia Sattar, Syed Abid Ali, Mustafa Kamal, Aftab Ahmed Khan, and Atiya Abbasi*

International Centre for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

Received 1 December 2003

Abstract

Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin.

These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against

the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families

Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an

ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from

L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin

inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI

shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of

172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a

disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI

and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We

have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or

complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique

features of LTI.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Leucaena leucocephala; Leguminosae; Kunitz inhibitor; Soybean trypsin inhibitor; Homology modelling; Three-dimensional structure

predictions

Proteinaceous inhibitors interact reversibly with

proteinases forming stoichiometric complexes and

competitively influence the catalytic activity of these

enzymes. Multiple molecular forms of these proteinshave been characterized from microorganisms, plants,

and animals. These proteins have long been considered

as anti-nutritional factors and implicated in various

physiological functions such as regulation of proteolytic

cascades, safe storage of proteins as well as defense

molecules against plant pests and pathogens [1]. Pro-

teinaceous inhibitors have been isolated and character-

ized from a variety of plants, most notably from legumes

* Corresponding author. Fax: +92-21-924-3190.

E-mail address: [email protected] (A. Abbasi).

0006-291X/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2003.12.177

[2]. The realization that these proteins might have im-

portant roles as defensive agents provided the basic

stimulus for research related to structure revealing a

number of interesting interrelationships. Interest in en-zyme inhibitors from plants began in the 1940s, when

Kunitz [3,4] isolated and purified a heat labile protein

from soybean, which inhibited trypsin. Later Robert

et al. [5] found an inhibitor of a-amylase in the grains of

many cereals. The possibility that such proteins could

constitute a human health hazard quickly led to many

studies to determine the extent of distribution in the

plant. The best known groups from seeds include in-hibitors of serine proteinases (EC.3.4.21) such as trypsin

(EC.3.4.21.4), chymotrypsin (EC.3.4.21.3), and subtili-

sin. Numerous examples are also known for inhibitors

mail to: [email protected]

756 R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765

of cysteine proteases (EC.3.4.22), aspartyl proteases(EC.3.4.23), and metallo-protease (EC.3.4.12).

Kunitz type serine proteinase inhibitors are found in

large quantity in the seeds of Leguminosae subfamilies,

i.e., Mimosoideae, Caesalpinoideae, and Papilionoideae.

Kunitz type inhibitors normally occur as single poly-

peptide chains however inhibitors from the subfamily

Mimosoideae have been shown to be dimeric proteins.

Some of the serine proteinase inhibitors have also beenfound to initiate platelet aggregation, blood coagula-

tion, fibrinolysis, and inflammation. In view of the

ability of the inhibitors to block enzymes, plant Kunitz

inhibitors have found use as tools in the study of bio-

chemical processes [6].

Trypsin inhibitor (LTI) belongs to a leguminous

plant Leucaena leucocephala (subfamily Mimosoideae).

Sequence analysis shows that LTI belongs to the familyof Kunitz soybean trypsin inhibitor (STI). Biochemical

studies show that LTI blocks enzymes involved in blood

clotting and fibrinolysis, has anti-inflammatory effects,

and decreases bradykinin release. Multiple mechanisms

are involved in the pathological changes that cause ac-

tivation of several pathways of inflammation and due to

the complex interactions between the various compo-

nents of these systems. In the present study we haveconstructed the three-dimensional structure of LTI and

predict its possible modes of interaction with trypsin.

We also predict some functional motifs such as phos-

phorylation and myristoylation in the three-dimensional

structure of LTI. These studies are expected to provide a

basic understanding of its interactions with different

molecules.

Methods

Primary sequences of LTI were obtained from Swiss Protein Data

Bank [7]. The Programs FASTA [8] and BLAST [9] were employed

for detecting similarities between sequences. Multiple sequence

alignments of LTI and its related sequences were carried out by

CLUSTAL X program with default parameters and finally, the

multiple alignments were manually adjusted if necessary. The coor-

dinates of STI and STI:trypsin complex were obtained by Brookha-

ven Protein data bank [10]. The secondary structure predictions of

LTI were carried out using the PHD method (http://www.embl-he-

idleberg.de/predictprotein/). The three-dimensional model of LTI was

constructed using the crystal structural coordinates of STI complex

with PPT in two crystal forms (PDB id: 1avw.pdb, 1avx.pdb) known

at a resolution of 1.75 and 1.9�A [11,12], respectively. Automated

homology model building was performed using protein structure

modelling program MODELLER 6 [13,14] which models protein

tertiary structures by satisfaction of spatial restraints. The input for

the program MODELLER consisted of the aligned sequence of LTI

and STI, and a steering file which gives all the necessary commands to

the Modeller for generating the homology models of the LTI on the

basis of its alignment with the crystal coordinates of the template.

Many runs of model building were carried out in order to obtain the

most plausible model.

The evaluation of the predicted LTI model, i.e., analysis of ge-

ometry, stereochemistry, and energy distributions in the models, was

performed using either the ENERGY commands of MODELLER or

using Programs “PROCHECK” and Whatcheck [15,16]. In addition,

the variability in the predicted model, i.e., RMSD was calculated by

superposition of Ca traces and backbones onto the template crystal

structure. The protein structures were visualized and analyzed on SPD

viewer (3.7), WebLab Viewer (4.0), and RASMOL (2.6). Conforma-

tional changes observed upon binding of ligands, i.e., /=w angles were

calculated for active site residues (P3, P2, P1, P10, P20, and P30) for the

free inhibitor as well as the enzyme inhibitor complex. In order to

obtain a plausible prediction with respect to interactions of the

inhibitor with corresponding subsite, e.g., S1 in the enzyme, all con-

served water molecules in experimental structure were also included in

the modelling procedure.

Results and discussion

Sequence analysis

Multiple sequence alignment of soybean-like trypsin

inhibitor (Fig. 1A) family (retrieved from Pfam: http://

pfam.wustl.edu/) reveals variable levels of sequence

similarity. Accordingly the family can be divided intothree major groups, i.e., trypsin-like, chymotrypsin-like,

and subtilisin-like Kunitz type inhibitors based on the

P1 specificity. Trypsin-like inhibitors are identified as

having basic amino acid residues at active site, i.e., Lys/

Arg (e.g., at residues Arg62 in LTI). In contrast, resi-

dues such as Phe, Leu, Tyr, and Met at this position are

typical for chymotrypsin inhibitor [17]. The nature of

position P1 determining the primary specificity (P1) ofthese inhibitors is highly conserved. However, the resi-

dues at neighboring positions (P20, P30) show consider-

able variations as shown in multiple sequence alignment.

A large number of sequences are available in Swiss

Protein data bank whereas three-dimensional structural

information is available only for some of these inhibi-

tors.

The phylogenetic distribution (Fig. 1B) analysis ofplant proteinase inhibitor family shows that these in-

hibitors can be further divided into subclasses such as

Kunitz type trypsin/chymotrypsin inhibitor, double

headed Bowman Birk inhibitor, Kazal inhibitors, PI-1,

etc. Among these the Kunitz-inhibitor family can be

divided into five subclasses on the basis of chain length.

The L. leucocephala trypsin inhibitor (LTI) contains 172

amino acid residues (Fig. 2). The protein showed thehighest homology with STI (soybean trypsin inhibitor,

39%) with two conserved disulfide bonds (according to

LTI C38–C83 and C130–C141).

Predicted three-dimensional structure of LTI

LTI is made up of two polypeptide subunits desig-

nated as a and b containing 137 and 37 residues,

respectively. The two chains interact with each other

and form a compact structure possessing features char-

acteristic of Kunitz type trypsin inhibitor as shown in

http://www.embl-heidleberg.de/predictprotein/

http://www.embl-heidleberg.de/predictprotein/

http://pfam.wustl.edu/

http://pfam.wustl.edu/

Fig. 1. (A) Multiple Sequence alignment of Kunitz type trypsin inhibitor, sequences are retrieved from http://smart.embl-heidelberg.de/. (B) Phy-

logenic tree derived for LTI related sequences isolated so far. Phylogenetic analysis of these sequences using the neighbor joining distances method

within Phylip package.

R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765 757

Fig. 2. Multiple sequence alignment of template STI (1avx and 1avw) and target (LTI). LTI has threefold internal symmetry. b-Sheet in this subunit is

labeled as An, Bn, and Cn where n indicates the number of sheets. Conserved cysteine residues are shown in blue and line represents the disulfide bond

between them. Conserved residues are marked as (*).


Fig. 3A. In spite of the dimeric nature LTI shows similar

overall geometry as seen for the monomeric inhibitors of

the STI family. The inhibitor possesses four cysteine

residues leading to two disulfide bridges, one betweenCys40 and Cys86 in the a chain and the other between

Cys 132 and Cys143 in the b-chain. Both the disulfide

bond and hydrogen bonds (Table 1) serve to increase the

stability of the predicted structure. The reactive site

(Arg62–Leu64) protrudes out from the loop of the achains. Studies carried out so far indicate that only in-

hibitors from this primitive subfamily are formed by two

chains which arise as a result of proteolytic cleavage of asusceptible bond in the single precursor, whereas all

other inhibitors from the papilionoidea and caesalpi-

noidea subfamilies are single polypeptide chain proteins

[18].

Schematic representation of the predicted model of

LTI–PPT complex consists of 12 anti-parallel b-strandsand a long loop connecting the b-strands. As seen in the

crystal structure of STI, six of the strands are arrangedin an anti-parallel b-barrel with the top six strands

forming the lid, symmetrically arranged in three pairs

around the barrel axis (Fig. 3A).

The structure of LTI displays threefold internal

symmetry (represented as A–C chains) as seen in STI.

The repeating unit is a four-stranded motif consisting of

Ca atoms between the complexed STI and LTI models.

In each unit amino acids are structurally organized as

L-b1, L-b2, L-b3, and L-b4 where L denotes a loop

connecting consecutive b-strands. Superposition of these

domains shows high degree of similarities for the bstrands but not for the connecting loops. Strands b1 andb4 from the same subdomain are adjacent, whereas

strand b1 is hydrogen bonded to the strand b4 of the

previous domain and strand b4 is hydrogen bonded to

the strand b1 of the following one. The N-terminus, the

loop between A4 and B1, and the shorter loop between

B4 and C1 lie at the bottom of the barrel. This type of

topology has been described as b trefoil and its struc-

tural determinants have been analyzed [19,20].The assignment of secondary structure elements is

presented in Table 1. The interaction between strands

from different domains (A4–B1, B4–C1, and C4–A1) are

much stronger than the intra-subdomain contacts (A1–

A4, B1–B4, and C1–C4). In the C subdomain, the ex-

posed side of the first strand (C1) maintains, through

hydrogen bond, a regular b-conformation to the begin-

ning of the C2 strands; a weaker interaction between B1and B2 is also observed. The basic hydrogen-bonding

pattern is the anti-parallel ladder between the second

and third strands (b2 and b3 of the same domain).

Evaluation of models

Procheck summary of LTI shows that 81.3% residues

are in the most favorable region, 13.2% in the allowed

Fig. 3. (A) Schematic representation of the predicted model of LTI showing the arrangements of a-helices, b-sheets, and loop regions in the predicted

three-dimensional structures of LTI. LTI is a dipeptidyl trypsin inhibitor. Small subunit is presented in blue color. (For interpretation of the ref-

erences to color in this figure legend, the reader is referred to the web version of this paper.) (B(I)) Structural superposition of homology model of

LTI with the crystal structure of STI. RMS deviation between the two structures was only 0.58�A2. (II) LTI possess threefold internal symmetry. The

picture depicted the structural superposition of homology model of LTI subunits (A–C).


region, and 4.9% in the generously allowed region andonly one residue (Thr98) is in the disallowed region. This

residue is however not present near the active site and as

such is not expected to affect the LTI predicted structure.

Structural differences between crystal structure of STI

and predicted three-dimensional structure of LTI:PPT

models were calculated by superimposing both struc-

tures (Fig. 3B). The rmsd values between the crystal

structure of STI:PPT (ortho and tetra) and homologymodel of LTI:PPT calculated for Ca traces and main

chain atom were 0.58 and 0.47�A, respectively. The rmsd

values and small variability among STI models and ex-

perimental structures reflect the presence of strong re-

straints in most regions and emphasize a similar folding

pattern among these inhibitors.

Binding pattern of LTI with trypsin

LTI belong to the family of substrate-like inhibitors,

which possess an exposed reactive site loop as shown in

Fig. 4. Unlike other inhibitors this loop is not con-

strained by the secondary structural elements and di-

sulfide bridges in the LTI molecule and as such is not

expected to limit its conformation freedom.

The reactive site loop containing the scissile bondArg62–Ile63 is located at the bottom of the molecule

between strand A4 and B1 which belong to the b-barrel.In spite of the structural similarity, there is a small

change in the orientation of P1 (Arg62) site (Fig. 5A).

Also some minor differences exist in the interaction

pattern between orthorhombic and tetragonal crystal

Table 1

Secondary structure distribution of LTI

~b-Strands Loops Disulfide

bond

N-terminal 1–12

A1 13–19

A1–A2 20–28

A2 29–31

A2–A3 32–40 Cys38

A3 41–45

A3–A4 46–54

A4 55–58

A4–B1 59–70 Reactive

site (62–64)

B1 71–72

B1–B2 73–87 Cys83

B2 88–93

B2–B3 94–99

B3 100–103

B3–B4 104–113

B4 114–120

B4–C1 121–128 Cys130

C1 129–131

C1–C2 132–143 Cys140

C2 144–147

C2–C3 148–153

C3 154–158

C3–C4 159–165

C4 166–169


structures of the STI complex itself. Thus, 12 amino acid

residues in the STI molecule interact with PPT in the

orthorhombic crystal structure. These include Asp1,

Fig. 4. Shows the LTI–trypsin complex model. The inhibitor backbone is

Residues present at different positions (P1, P2, P3, P10, P20, and P30) of the pri

Phe2, Asn13, Pro61 (P3), Tyr62 (P2), Arg63 (P1), Ile64(P10), Arg65 (P20), His71, Pro72, Trp117, and Arg119.

In the tetragonal crystal structure, the three residues

His71, Pro72, and Arg119 do not interact with PPT. In

the LTI structure eight residues (Asp139, Asn136,

Pro60, Tyr61, Arg62, Ile63, and Leu64) are in con-

tact with PPT. This change is mainly due to the

conformational changes observed at the reactive site

surface. However, the pattern of hydrogen bonding in-teraction involving the reactive site loop residues from

Pro (P3) to Ile (P10) is well conserved between the two

structures, i.e., STI:PPT and predicted model of

LTI:PPT. Also in the tetragonal structure 10 of the 11

sites show similar interaction. Thirteen hydrogen bonds

are involved in the interaction between LTI and PPT.

LTI binds with trypsin at two sites, one is the central

reactive site (P3, P2, P1, P10, P20, and P30) and the otheris through side chains of Asn136 and Asp139.

Geometry of the reactive site of carbonyl group

The geometry of the carbonyl group at the P1 posi-tion is of great importance in generating an interaction

between the inhibitor and proteinase during catalysis.

Thus, the carbonyl carbon in LTI interacts by forming a

hydrogen bond with NH of Ser195 of trypsin in both

STI:PPT (ortho) and STI:PPT (tetra) models, the dis-

tances between Arg62 and Ser195 being 2.76 and 2.71�A,

respectively. Also, the carbonyl carbon atom in STI was

found to be within the van der Waals distance from

shown as solid ribbon and protease backbone is shown as Ca-traces.

ncipal binding loop of LTI are depicted as ball and stick representation.

Fig. 5. (A) Stereoview of the Ca-atom backbone of STI (a) and LTI (b) showing the different orientations of the reactive site. The positions of Arg63

and Asn13 are shown. (B) Interaction between Arg62 (P1) of LTI and S1 residues of trypsin (shown in pink color). (For interpretation of the

references to color in this figure legend, the reader is referred to the web version of this paper.)


Ser195. Fig. 5B shows the geometry and mode of in-

teraction of Arg62 (P1).

Conformation of the reactive site of LTI–trypsin complex

Changes in the reactive site of LTI were calculated by

superposition of free LTI with complex LTI:PPT. The

two structures, i.e., STI and BPTI show a large devia-

tion between the P4 and P20 angles. Despite the signifi-

cant differences in the main chain conformational angles

at P20 position (arginine) for STI and BPTI, bothguanidinium groups point in the same direction as seen

in STI and MCTI. A large deviation is observed for

angles of P3 and P20 whereas ETI (22, 23, 24: Erythrina

caffra trypsin inhibitor) and WCI (25: winged bean

chymotrypsin inhibitor) show similar conformations as

STI at P20. The reactive site loops of Kunitz (BPTI),

Kazal, Squash, and PI-1 families are constrained by

disulfide bridges limiting the conformational freedom.

However, no disulfide bridge is adjacent to the reactivesite loop in LTI or STI. Furthermore, the reactive site

loop of LTI is held in a favorable conformation sup-

ported by hydrogen bonds. The predicted model of

LTI–PPT complex however shows a different / angle for

Arg62 in the LTI:PPT complex as compared to STI:PPT

whereas w angles for both LTI–PPT and STI–PPT

complexes are similar. Similarly the active site residues

of LTI–PPT and STI–PPT complexes show wide

Table 2

Comparison of dihedral angles (/=w) of reactive site loop residues of trypsin inhibitor (ortho and tetra) [11,12] (Blow), Sweet et al. (1974); ETI, [28];

WCI, Dattagupta et al., Wlodawer et al. (1987); BPTI, [29]; MCTI, Huang et al. (1993)

P4 P3 P2 P1 P10 P20 P30 P40

STI S P Y R I R F I

Free LTI S P Y R I L I G

Free STI )112/106.4 )70/)28 )62/158 )84/21 )60/148 )90/)22 )127/126STI:PPT (ortho) )114/144 )58/)34 )56/139 )89/38 )83/148 )67/)38 )118/155STI:PPT (tetra) )126/149 )67/)23 )65/121 )70/10 )59/165 )88/)29 )115/170Free LTI )99.5/)154.6 )154.6/)69.7 )97.1/179.3 )103.9/10.3 )73.3/166.6 70.8/)17.0 )13.0/157.2LTI:PPT )99.5/126.3 )154.5/)69.7 )97.2/179.4 )104/10.3 )73.3/166.5 )70.8/17 )130/157.2


differences in /=w angles of P2, P3, and P4 site residues

but small difference in /=w angles of P10 and P30 site

residues.

The back bone conformation of the reactive site loop

of LTI does not change significantly upon complexation

with PPT. Table 2 shows that / and w angles of free LTI

and LTI:PPT are equal. Also, despite the lack of disul-

fide bridges or strong electrostatic interactions, thereactive site loop is maintained in a well-ordered con-

formation by a network of hydrogen bonding involving

the N-terminal loop (residue 1–14).

In LTI the side chain of Asn136, which is not part of

binding segments, plays an important role in stabilizing

the reactive site loop conformation through a network

of hydrogen bonds. Similar stabilization is also observed

in ETI and WCI.

Characteristics of the S1 subsite

The S1–P1 binding site is the primary specificity de-

terminant for serine proteinases. In trypsin, the S1subsite is a well-defined pocket formed by the residues of

the segments 189–95 and 214–220. The main chain and

several of the side chains of these residues and many

ordered water molecules present at this site make an

extensive network of hydrogen bonding interactions

with the P1 (Arg62) side chain of LTI (Fig. 5B). The P1

residue Arg62 in STI–trypsin models extends directly

into this pocket. The three “SP3” hydrogens of NE arein a staggered conformation allowing the NH2 group to

form two hydrogen bonds with the side chain of Asp189

(OD1 and OD2) and water molecules in the S1 pocket.

Additionally, the NH1 can form two hydrogen bonds

with main chain (carbonyl) atoms of Gly219 and water

molecules in the S1 pocket.

In the predicted model LTI–trypsin complex, NH1 of

Arg62 (P1) forms a hydrogen bond with water moleculeW801, which interacts further with another water mol-

ecule W809 through hydrogen bonding, which in turn is

hydrogen bonded to the carbonyl oxygen of Tyr228 of

the trypsin molecule. This hydrogen bonding network,

which extends via water molecules from LTI Arg62 side

chain to the main chain of residues in proteases (trypsin)

at the deeper end of the S1 binding site, has been de-

scribed as a structural motif with several water mole-

cules forming a channel [21–23]. Earlier, Bartunik et al.

identified a water channel in trypsin extending from the

specificity pocket to the surface of the enzyme. Sreeni-

vasan and Axelsen [22] identified 16 internal water sites

as being conserved in trypsin, trypsinogen, chymotryp-

sin and chymotrypsinogen, elastase, kallikrein, tonin,

and mast cell protease. This type of buried water mol-ecules has also been located in other serine proteases

such as thrombin [24] and subtilisin [25] and appears to

play a structural role. The location of buried water

molecule in the trypsin-like proteases is more extensive

[21]. The guanidinium group of Arg62 makes an ionic

interaction with the carboxylate ion of Asp189 in PPT.

Beside these interactions, two hydrogen bonds could be

formed in the LTI–trypsin complex model betweenArg62 NE and the main chain and side chain of Ser190.

The other interaction occurring between Arg62 of LTI

and main chain and side chain of trypsin is shown in

Fig. 5B.

The interactions observed in the LTI–trypsin complex

in particular the Arg (P1) interaction with S1 subsite

appear to be mainly due to the modification observed in

the vicinity. The extensive literature dealing with sub-strate specificity shows clearly that even though trypsin

has very strong preferences for a basic side chain at its

primary specificity site P1, the secondary specificity also

plays an important role in substrate/inhibitor bindings

[26–28]. Residues present at different secondary speci-

ficity sites can either influence the inhibitor directly by

interacting with the respective subsites at the enzyme

surface or affect the interactions of the P2 residues withits binding pocket. Thus, the difference in secondary

specificity of LTI compared to other Kunitz type in-

hibitors around Arg62 in the LTI–trypsin complex

model leads to a different mode of interaction observed

at the S1 binding site.

The Leu at position 64 present in LTI binds to the S20

subsite while in STI this position is occupied by Arg in

STI. Like S1 binding site, this subsite appears to beequally active in binding. The protease surface at this

subsite is composed of residues Ser39, His40, Gln192,

and Gly193 of the protease with Phe41, Gly66, Asn11,

and Tyr10 of the inhibitor contributing to the formation


of a hydrophobic subsite cleft. The multiple alignmentsof Kunitz type inhibitor show that all hydrophobic

residues are reasonably well conserved at this position.

Leu64 in LTI–trypsin has also a solvent exposed con-

formation similar to that seen in STI. The carboxylic

group of Leu64 forms a hydrogen bond with water

molecule W840. This pocket is smaller in trypsin as

compared to chymotrypsin because of the larger volume

of the subsite, i.e., tyrosine 151. In chymostrysin thisposition is occupied by threonine [29].

Phosphorylation and myristoylation

Protein kinases play a vital role in regulating andcoordinating aspects of metabolism, gene expression,

cell mortality, cell differentiation, cell division, and

protein activity by “switching on and off” mechanism

allowing phosphorylation and dephosphorylation to

proceed in an orderly fashion in cellular life.

Some functional motifs could be picked up in LTI by

the “PROSITE Search” indicating the possible phos-

phorylation site in the structure. The structural motifcorrelates well with the pattern recognized by protein

kinase C and casein kinase II. In LTI two sequence

patches, i.e., Thr–Ser–Arg (position 49–51) and Ser–

Ser–Arg (position 87–89) have been identified as possi-

ble targets for protein kinase C phosphorylation, with

Thr49 and Ser87 being the most probable phosphory-

lation sites.

In LTI, a possible myristoylation site, i.e., Gly–Leu–Glu–Leu–Ala–Arg is seen at position 27–32. The myri-

stoylated residue can bind with viral proteins and

appears to play a purely structural role. In addition to

mediating protein–protein interactions, protein myri-

stoylation is also known to enhance protein–lipid

interactions by targeting and binding polypeptide chains

to different types of membranes. Studies on various

proteins indicate that interaction of STI with the non-phosphorylated form of the protein can lead to an inac-

tive complex. Thus, the interaction of STI with Abelson

tyrosine kinases (Abl) has been shown to be helpful in

preventing inadvertent activation of protein kinase (Abl)

thereby controlling chronic myelogenous leukemia

CML. Thus, STI offers a very good prospect for the de-

velopment of specific protein kinase inhibitor [30].

Conclusion

Serine proteinase inhibitors have been reported from

a number of plants and have been found to vary both in

sequence and structure. Trypsin inhibitor LTI, from L.

leucocephala, is a dimeric proteinase inhibitor belonging

to the Kunitz family. At present no information isavailable on this type of Kunitz inhibitors. LTI consists

of 172 amino acid residues with two disulfide bridges

which provide stability to the structure. N-terminal se-quence of LTI shows a high degree of homology with

proteinase inhibitors which have been shown to function

as defense arsenals against attack by insects [31].

Structural comparison of LTI and STI shows that LTI is

a dipeptidyl inhibitor whereas STI is a monomeric

protein. However, it can be speculated that the inhibi-

tors have originated from the same precursor which is

proteolytically cleaved to give the dipeptide. In LTIboth chains are linked by disulfide bridges and combine

to give a slightly pear-shaped inhibitor. From the above

structural discussion of homology model of LTI:PPT

complex we conclude that LTI belong to the family of

substrate-like inhibitors, which possess an exposed re-

active site loop. In contrast to other inhibitors, the re-

active site loop of LTI is not constrained by secondary

structural elements and disulfide bridges thus allowingconformational freedom. The backbone conformation

of the reactive site loop of LTI does not change signif-

icantly upon complexation with trypsin indicating that

the reactive site of LTI is stable despite the absence of a

disulfide bridge near the active site. Comparison of the

active site residues shows that P1–P4 positions of LTI

are homologous to STI however the P20 to P40 positions

show wide variations. It has been observed earlier thatdespite structural variations, active site residues in this

class of inhibitors are mostly conserved. P1 site is Arg

which tends to inhibit trypsin-like protease activity. In

LTI its scissile site P1–P10 (Arg–Ile) is conserved. The P1

residues of Arg62 make the most extensive hydrogen

bonds with PPT. The side chain of Arg62 occupies its

expected position in the primary binding pocket of PPT.

The guanidinium group of Arg62 makes an ionic inter-action with the carboxylate group of Asp189 in PPT.

The phenolic side chain of Tyr61 (P2) is positioned be-

tween the side chain of Leu99 and His57, being parallel

to the imidazole ring of the latter and its hydroxyl group

forms a hydrogen bond with the carbonyl “O” atom of

Gly96.

The major contribution in the binding of LTI with

the protease molecule comes from eight residues (Thr98,Gln117, Pro60, Tyr61, Arg62, Ile63, and Leu64).

However, the pattern for the hydrogen bonding inter-

action involving the reactive site loop residues from Pro

(P3) to Ile (P10) is well conserved between the crystal

structures STI:PPT and LTI:PPT. Another important

interaction which plays an important role in stabilizing

the enzyme:inhibitor complex arises from the side chain

of Asn136 which forms a network of hydrogen bondsaround the reactive site loop. Similar stabilization is also

observed for ETI (E. caffra trypsin inhibitor) and WCI

(winged bean chymotrypsin inhibitor), although the

hydrogen bonding patterns are not identical.

Serine proteinase inhibitors have found wide appli-

cation as therapeutic agents and are well suited for in-

hibition of many proteases, and indirectly for the


control of many diseases, e.g., cancer [32], Alzheimer’sdisease [33], etc. It has already been reported that LTI

participates in blood clotting and has been shown to

inhibit kinin release in vitro. Thus, intravenous admin-

istration of Kallikrein has been shown to decrease paw

edema induced by carrageenin or heat in male Wistar

rats. In addition, lower concentration of bradykinin was

found in limb perfusion fluids of LTI treated rats. De-

spite the important role played by these inhibitors, littleattempt has been made for the characterization of these

proteins. The predicted three-dimensional structure of

LTI and its interaction with trypsin indicate similar

binding affinity as seen for STI. Furthermore, analysis

of functional motifs in the LTI sequence, for example,

phosphorylation site indicates changes in conformation

which may influence the functioning of LTI. Similarly

myristoylation of LTI may influence protein–proteinand protein–lipid interactions. These functional motifs

also help in providing a basic understanding of the in-

teraction of LTI with other molecules, for example, STI

is known to bind with Abl and influence its phosphor-

ylation or inadvertent activation which in turn reduces

the risk of CML. Thus, the understanding of structural

characteristics of LTI may be useful not only in treating

pathological conditions but also possesses the biotech-nological potential for use as an agent against phy-

tophagous insects.

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