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Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling

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Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling
  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 againstthe attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the familiesLeguminosae, Graminae, and Solanaceae.  Leucaena leucocephala  belongs to the family Leguminosae. It is widely used both as anornamental 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 trypsininhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTIshows 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 adisulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STIand LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. Wehave also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity orcomplexation with other molecules. Some of the differences observed at the active site and functional sites can explain the uniquefeatures of LTI.   2003 Elsevier Inc. All rights reserved. Keywords: Leucaena leucocephala ; Leguminosae; Kunitz inhibitor; Soybean trypsin inhibitor; Homology modelling; Three-dimensional structurepredictions Proteinaceous inhibitors interact reversibly withproteinases forming stoichiometric complexes andcompetitively influence the catalytic activity of theseenzymes. Multiple molecular forms of these proteinshave been characterized from microorganisms, plants,and animals. These proteins have long been consideredas anti-nutritional factors and implicated in variousphysiological functions such as regulation of proteolyticcascades, safe storage of proteins as well as defensemolecules against plant pests and pathogens [1]. Pro-teinaceous inhibitors have been isolated and character-ized from a variety of plants, most notably from legumes[2]. The realization that these proteins might have im-portant roles as defensive agents provided the basicstimulus for research related to structure revealing anumber of interesting interrelationships. Interest in en-zyme inhibitors from plants began in the 1940s, whenKunitz [3,4] isolated and purified a heat labile proteinfrom soybean, which inhibited trypsin. Later Robertet al. [5] found an inhibitor of   a -amylase in the grains of many cereals. The possibility that such proteins couldconstitute a human health hazard quickly led to manystudies to determine the extent of distribution in theplant. The best known groups from seeds include in-hibitors of serine proteinases (EC.3.4.21) such as trypsin(EC., chymotrypsin (EC., and subtili-sin. Numerous examples are also known for inhibitors * Corresponding author. Fax: +92-21-924-3190. E-mail address: (A. Abbasi).0006-291X/$ - see front matter    2003 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2003.12.177Biochemical and Biophysical Research Communications 314 (2004) 755–765 BBRC  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 inlarge 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 subfamilyMimosoideae 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 theability of the inhibitors to block enzymes, plant Kunitzinhibitors have found use as tools in the study of bio-chemical processes [6].Trypsin inhibitor (LTI) belongs to a leguminousplant  Leucaena leucocephala  (subfamily Mimosoideae).Sequence analysis shows that LTI belongs to the familyof Kunitz soybean trypsin inhibitor (STI). Biochemicalstudies show that LTI blocks enzymes involved in bloodclotting and fibrinolysis, has anti-inflammatory effects,and decreases bradykinin release. Multiple mechanismsare involved in the pathological changes that cause ac-tivation of several pathways of inflammation and due tothe complex interactions between the various compo-nents of these systems. In the present study we haveconstructed the three-dimensional structure of LTI andpredict its possible modes of interaction with trypsin.We also predict some functional motifs such as phos-phorylation and myristoylation in the three-dimensionalstructure of LTI. These studies are expected to provide abasic understanding of its interactions with differentmolecules. Methods Primary sequences of LTI were obtained from Swiss Protein DataBank [7]. The Programs FASTA [8] and BLAST [9] were employedfor detecting similarities between sequences. Multiple sequencealignments of LTI and its related sequences were carried out byCLUSTAL X program with default parameters and finally, themultiple 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 ( The three-dimensional model of LTI wasconstructed using the crystal structural coordinates of STI complexwith PPT in two crystal forms (PDB id: 1avw.pdb, 1avx.pdb) knownat a resolution of 1.75 and 1.9  A [11,12], respectively. Automatedhomology model building was performed using protein structuremodelling program MODELLER 6 [13,14] which models proteintertiary structures by satisfaction of spatial restraints. The input forthe program MODELLER consisted of the aligned sequence of LTIand STI, and a steering file which gives all the necessary commands tothe Modeller for generating the homology models of the LTI on thebasis of its alignment with the crystal coordinates of the template.Many runs of model building were carried out in order to obtain themost plausible model.The evaluation of the predicted LTI model, i.e., analysis of ge-ometry, stereochemistry, and energy distributions in the models, wasperformed using either the ENERGY commands of MODELLER orusing Programs “PROCHECK” and Whatcheck [15,16]. In addition,the variability in the predicted model, i.e., RMSD was calculated bysuperposition of C a  traces and backbones onto the template crystalstructure. The protein structures were visualized and analyzed on SPDviewer (3.7), WebLab Viewer (4.0), and RASMOL (2.6). Conforma-tional changes observed upon binding of ligands, i.e.,  / = w  angles werecalculated for active site residues (P3, P2, P1, P1 0 , P2 0 , and P3 0 ) for thefree inhibitor as well as the enzyme inhibitor complex. In order toobtain a plausible prediction with respect to interactions of theinhibitor with corresponding subsite, e.g., S1 in the enzyme, all con-served water molecules in experimental structure were also included inthe modelling procedure. Results and discussion Sequence analysis Multiple sequence alignment of soybean-like trypsininhibitor (Fig. 1A) family (retrieved from Pfam: reveals variable levels of sequencesimilarity. Accordingly the family can be divided intothree major groups, i.e., trypsin-like, chymotrypsin-like,and subtilisin-like Kunitz type inhibitors based on theP1 specificity. Trypsin-like inhibitors are identified ashaving 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 aretypical for chymotrypsin inhibitor [17]. The nature of position P1 determining the primary specificity (P1) of these inhibitors is highly conserved. However, the resi-dues at neighboring positions (P2 0 , P3 0 ) show consider-able variations as shown in multiple sequence alignment.A large number of sequences are available in SwissProtein data bank whereas three-dimensional structuralinformation is available only for some of these inhibi-tors.The phylogenetic distribution (Fig. 1B) analysis of plant proteinase inhibitor family shows that these in-hibitors can be further divided into subclasses such asKunitz type trypsin/chymotrypsin inhibitor, doubleheaded Bowman Birk inhibitor, Kazal inhibitors, PI-1,etc. Among these the Kunitz-inhibitor family can bedivided into five subclasses on the basis of chain length.The  L. leucocephala  trypsin inhibitor (LTI) contains 172amino acid residues (Fig. 2). The protein showed thehighest homology with STI (soybean trypsin inhibitor,39%) with two conserved disulfide bonds (according toLTI C 38  –C 83  and C 130  –C 141 ). 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 otherand form a compact structure possessing features char-acteristic of Kunitz type trypsin inhibitor as shown in 756  R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765  Fig. 1. (A) Multiple Sequence alignment of Kunitz type trypsin inhibitor, sequences are retrieved from (B) Phy-logenic tree derived for LTI related sequences isolated so far. Phylogenetic analysis of these sequences using the neighbor joining distances methodwithin Phylip package. R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765  757  Fig. 3A. In spite of the dimeric nature LTI shows similaroverall geometry as seen for the monomeric inhibitors of the STI family. The inhibitor possesses four cysteineresidues leading to two disulfide bridges, one betweenCys40 and Cys86 in the  a  chain and the other betweenCys 132 and Cys143 in the  b -chain. Both the disulfidebond and hydrogen bonds (Table 1) serve to increase thestability of the predicted structure. The reactive site(Arg62–Leu64) protrudes out from the loop of the  a chains. Studies carried out so far indicate that only in-hibitors from this primitive subfamily are formed by twochains which arise as a result of proteolytic cleavage of asusceptible bond in the single precursor, whereas allother 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 thecrystal structure of STI, six of the strands are arrangedin an anti-parallel  b -barrel with the top six strandsforming the lid, symmetrically arranged in three pairsaround the barrel axis (Fig. 3A).The structure of LTI displays threefold internalsymmetry (represented as A–C chains) as seen in STI.The repeating unit is a four-stranded motif consisting of C a  atoms between the complexed STI and LTI models.In each unit amino acids are structurally organized asL- b 1 , L- b 2 , L- b 3 , and L- b 4  where L denotes a loopconnecting consecutive  b -strands. Superposition of thesedomains shows high degree of similarities for the  b strands but not for the connecting loops. Strands  b 1  and b 4  from the same subdomain are adjacent, whereasstrand  b 1  is hydrogen bonded to the strand  b 4  of theprevious domain and strand  b 4  is hydrogen bonded tothe strand  b 1  of the following one. The N-terminus, theloop between A4 and B1, and the shorter loop betweenB4 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 ispresented in Table 1. The interaction between strandsfrom different domains (A4–B1, B4–C1, and C4–A1) aremuch 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, throughhydrogen 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-bondingpattern is the anti-parallel ladder between the secondand third strands ( b 2  and  b 3  of the same domain). Evaluation of models Procheck summary of LTI shows that 81.3% residuesare in the most favorable region, 13.2% in the allowed Fig. 2. Multiple sequence alignment of template STI (1avx and 1avw) and target (LTI). LTI has threefold internal symmetry.  b -Sheet in this subunit islabeled as A n , B n , and C n  where  n  indicates the number of sheets. Conserved cysteine residues are shown in blue and line represents the disulfide bondbetween them. Conserved residues are marked as (*).758  R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765  region, and 4.9% in the generously allowed region andonly one residue (Thr98) is in the disallowed region. Thisresidue is however not present near the active site and assuch is not expected to affect the LTI predicted structure.Structural differences between crystal structure of STIand predicted three-dimensional structure of LTI:PPTmodels were calculated by superimposing both struc-tures (Fig. 3B). The rmsd values between the crystalstructure of STI:PPT (ortho and tetra) and homologymodel of LTI:PPT calculated for C a  traces and mainchain atom were 0.58 and 0.47  A, respectively. The rmsdvalues and small variability among STI models and ex-perimental structures reflect the presence of strong re-straints in most regions and emphasize a similar foldingpattern 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 inFig. 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 notexpected to limit its conformation freedom.The reactive site loop containing the scissile bondArg62–Ile63 is located at the bottom of the moleculebetween strand A4 and B1 which belong to the  b -barrel.In spite of the structural similarity, there is a smallchange in the orientation of P1 (Arg62) site (Fig. 5A).Also some minor differences exist in the interactionpattern between orthorhombic and tetragonal crystal Fig. 3. (A) Schematic representation of the predicted model of LTI showing the arrangements of   a -helices,  b -sheets, and loop regions in the predictedthree-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  A 2 . (II) LTI possess threefold internal symmetry. Thepicture depicted the structural superposition of homology model of LTI subunits (A–C). R. Sattar et al. / Biochemical and Biophysical Research Communications 314 (2004) 755–765  759
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