CHAPTER 7

THE PB28-13. 1 ANTIGEN-ANTIBODY

COMPLEX

Pb28 protein and therapeutic antibody 13.1

The Pb28-13.1 Antigen-Antibody Complex

7.1 Introduction Every year, 300-600 million people across the globe get affected by malaria and leading

to 1-3 million deaths [Snow et. al., 2005; World malaria rep0l1]. Plasmodium after

undergoing several stages of development inside human host forms gametocytes, which

enter mosquito midgut with blood meal and form micro and mega gametes. These

gametes fuse to form zygote which develops into motile ookinete and crosses the

mosquito midgut epithelium to form oocysts. Mature oocyst releases thousands of

sporozoites, which migrate to the salivary glands of mosquito to infect another human

host. Proteins of P28 family are present on ookinete surfaces of all known Plasmodium

species [Kumar et. aI., 1985; Vermeulen et. aI., 1985; Paton et. aI., 1993]. These proteins

start expressing immediately after fertilization and present on zygote, ookinete and young

oocyst stages of Plasmodium [Fries et. al., 1990]. These proteins are distributed evenly

and abundantly over the entire ookinete surface as shown by immunofluorescent antibody

staining [Winger et. aI., 1988] and immunogold electron microscopy [Sinden ct. aI.,

1987; Duffy et. aI., 1993]. Gene knock out studies indicate that P28 proteins play

important role in parasite survival inside mosquito midgut [Tomas et. aI., 2001]. Proteins

of P28 family contain a signal sequence; four EGF (epidermal growth factor) like

domains and a C-terminal GPI (glycosylphosphatidylinositol) anchor [Kaslow ct. aI.,

1988; Tsuboi et. aI., 1998]. It is well known that EGF-domains are present in surface

proteins participating in recognition, adhesion and signaling [Appell a et. aI., 1988],

which indicates that P28 proteins may have important roles in host parasite interactions.

Also P28 protein show limited sequence polymorphisms. It is presumably because these

proteins are not expressed in the vertebrate host and never exposed to the vertebrate

immune system [Carter et. al., 1989]. The process by which transmission blocking

antibodies inhibit parasite development is not clear. Results show that transmission

blocking antibodies arrest or slow down the movement of ookinete in the mosquito

midgut [Yoshida et. aI., 1999]. The levels of antibody produced were found proportional

with transmission blocking activity observed [Kaslow et. aI., 1996]. It has been shown if

ookinetes delayed in forming oocysts, it will not be able to survive in the harsh

proteolytic environment inside mosquito midgut [Malkin et. aI., 2005]. Recombinant

versions of transmission blocking antibodies also block transmission of parasite from

76

The Pb28-13.1 Antigen-Antibody Complex mosquito to human host [Tsuboi et. al., 1997; Han et. al., 2000]. The molecular structure

of scFv of 13.1 antibody bound to antigen Pb28 (Swissprot id Q04620) from P. berghei

may help in understanding mechanism of transmission blocking.

Recently, Protein-protein docking is becoming potential and popular means of

predicting antibody-antigen complex structures particularly in cases, where antigen­

antibody complex crystals prove difficult to obtain [Shivasubramanian ct. af., 2006; Gray

et. al., 2006]. In some cases, atomic level accuracy is within reach. In this case we have

used ZDOCK and RDOCK programs for docking Pb28 with scFv of 13.1 antibody.

These programs have successfully recapitulated structures of many known protein­

protein complexes and produced high accuracy predictions for multiple protein-protein

targets in the CAPRI challenge [Wiehe et. aI., 2005]. These programs will yield accurate

structures if some biochemical information is available for the studied complex.

Homology modeling of Pb28 protein (Swissprot id Q04620) suggested that mature Pb28

protein is a triangular flat molecule. It has four EGF domains with maximum variations

present in EGF domain III and B loop of EGF domain II, when compared with template

Pvs25 (PBD id lz27). Single chain variable fragment (scFv) of 13.1 antibody was

modeled with the help of W AM server. Loops in the W AM model were refined with the

help of ModLoop program to ensure that the CDRs of the scFv are modeled correctly.

Docking of Pb28 protein model with scFv of 13.1 antibody showed that EGF domain II

of Pb28 interacts with scFv of 13.1 antibody in accordance to Spano et. al. in 1996. In P.

berghei, the transmission blocking antibody 13.1 was mapped using deletions and

overlapping peptides to the sequence GLEKAFVC on the B loop of EGF Domain 2 of

Pb28 protein [Spano et. aI., 1996].

7.2 Materials and Methods 7.2.1 Template selection

Single letter amino acid sequence of Pb28 protein was taken from Swiss-Prot (id

=Q04620) [Gasteiger et. aI., 2003] release 50.4(http://www.expasy.org!uniprot/Q04620).

In order to select suitable template for Pb28 protein, PSI-BLAST

(http://www.ncbi.nlm.nih.gov/BLAST/)[Altschulet.al. , 1993] was performed against

the PDB database (http://www.rcsb.org/pdb/Welcome.do. 51977 structures) [Berman ct.

77

The Pb28-13.1 Antigen-Antibody Complex aI., 2000] which resulted in a single significant hit (E value =0.001) i.e. Pvs25 protein

from Plasmodium vivax. It is the only suitable template in PDB database for modeling

Pb28 protein. In both template and target proteins, there are 20 conserved cysteines in the

oxidized form and forming ten disulphide bonds. These disulphide bonds are formed

between 1-3, 2-4 and 5-6 cysteines of EGF domain II and EGF domain III. In case of

EGF domain I, all four cysteines are conserved forming disulphide bonds among 2-4 and

5-6 cysteines, whereas in EGF-domain IV, only four out of six cysteines are conserved.

Sequence alignment with template protein Pvs25, was done with the help of ClustalX

program (http://www.embl.de/..-.Chenna/clustalldarwinJindex.html)[Chennaet.al. , 2003].

Sequence identity between the two proteins was calculated with the help of GraphAlign

(http://daIwin.nmsu.edu/cgi-binJgraph_align.cgi)[Spaldinget.al., 2004] and Ident and

Sim program (http://www.bioinformatics.org/sms2/ident_ sim.html) [Stothard ct. o/.,

2000].

7.2.2 Homology modeling

Pb28 sequence was obtained from Swissprot database were modeled using MODELLER

version 9vl. This software implements homology modeling of proteins by satisfaction of

spatial restraints [Sali ct. a/., 1993]. Hundred models were generated for Pb28 protein

with the help of Modeller. Modeller automatically derives restraints from known related

structures. The restraints include distances, angles, dihedral angles, pairs of dihedral

angles and some other spatial restraints. Bond and angle values are taken from

CHARMM-22 force field. 3D models are generated by molecular probability density

function optimization. X-ray crystallographic structure of Pvs25 from Plasmodium vivax

(PDB id lz27) [Saxena ct. aI., 2004; Saxena et. aI., 2004; Saxena ct. aI., 2006], was used

as template. To model the variable loops ModLoop program

(http://alto.compbio.ucsf.edu/modloopllmodloop.html)wasused[Fiseret.al., 2000; Fiser

ct. aI., 2003]. B loop of EGF domain II was modeled with caution. Modeling of the scFv

of 13.1 antibody (NCBI accession no. JC581O) was done using Web Antibody Modeling

server: WAM (http://www.bath.ac.uk/cpad/)[Whitelegget.al..2000;Whiteleggct.al .•

2004] and refined with the help of Modeller and ModLoop.

78

The Pb28-13.1 Antigen-Antibody Complex 7.2.3 Model evaluation

Evaluation of the models of Pb28 protein were carried out using ProSA-web

(https:llprosa.services.came.sbg.ac.at/prosa.php) [SippI et. at., 1993; Wiederstein ct. aI.,

2007], PROCHECK (http://nihserver.mbi.ucla.edu/SAVS/)[Laskowaskiet.at. , 1993]

and WHATIF programs (http://swift.cmbi.kun.nIlWIWWWII)[Vriendet.al., 1990].

Only those models were selected, which showed satisfactory PROSA, PROCHECK and

WHATIF profile [Hooft et. aI., 1996; Morris et. aI., 1992]. To model the flexible loops,

ModLoop (http://alto.compbio.ucsf.edulmodloopllmodloop.html) was used. 3D structural

superimposition for comparing the structures was done by using program STAMP, which

is a part ofVMD version 1.8.4 [Humphrey et. aI., 1996].

7.2.4 Docking

ZDOCK (v2.1) program was used to predict the antigen-antibody complex from the

unbound proteins. ZDOCK (http://zlab.bu.edulzdock!) is an initial stage protein-protein

docking program, which employs Fast Fourier Transform algorithm to translate and

rotate the antigen around the surface of the antibody. ZDOCK evaluates pairwise shape

complementarity, desolvation and electrostatic energies [Chen ct. aI., 2003]. Script

"Mark sur" was used to mark the model PDB files in order to make the files readable to

ZDOCK program. Rigid body docking was done using ZDOCK and 2000 model

complexes were generated. Refinement and ranking of the obtained models was done

with the help of RDOCK (http://zlab.bu.edulzdock!)program[Liet.al., 2003]. RDOCK

program makes significant improvements upon top ZDOCK predictions by a three-stage

energy minimization using CHARMM program. RDOCK refines complex structures,

evaluates binding free energies and reranks complexes generated by ZDOCK program.

The script "rdock.pl" was used which uses CHARMM (v32) program [Brooks ct. aI.,

1983] for energy minimization. RDOCK minimizes the complexes generated by ZDOCK

by a process involving 3 steps: Step 1, Small clashes are removed to allow small

conformational changes; Step 2, Polar interactions are optimized and Step 3,

Optimization of charged interactions. In these steps, the charges were turned off for ionic

residues in step 1 and 2. To rerank the top RDOCK predictions, script

"rerank.pl" was used. A reranked list of complexes was obtained based on electrostatic,

79

The Pb28-13.1 Antigen-Antibody Complex vander Waals and Atomic contact energy (ACE) and top 30 complexes were selected out

of the 2000 solutions obtained. These top 30 complexes were divided into four different

groups based on their interaction with the EGF domain lor II or III or IV. Complexes

showing binding to B loop of EGF domain II, were selected and looked for antigen

antibody interaction. Complexes showing interaction with CDRs of the antibody were

selected and buried surface areas of these complexes were calculated using POPScomp

server (http://mathbio.nimr.mrc.ac.ukltoollpopscomp/)[Cavalloet.al. , 2003]. The

complex showing maximum buried surface area in the group was considered as the best

possible interaction between the two proteins. Residue-residue interactions for this

antigen-antibody complex were calculated usmg PDBsum server

(http://www.ebi.ac.uklthomton-srv/databases/pdbsum/upload.html)[Laskowaskiet.al. ,

2001].

7.3 Results and Discussion 7.3.1 Pb28 Protein and sequence conservation with Pvs25:

The Pb28 protein located on the surface of ookinete is considered promising malaria

transmission blocking vaccine candidate. Sequence of this P28 protein from P. berghei

anka strain was taken from Swiss-Prot release 50.4, 51977 entries

(http://au.expasy.org/sprot/). Fig. 7.6.1 depicts the multiple sequence alignment of Pb28

protein (Swiss-Prot id=Q04620) with the template protein Pvs25 (PDB id 1z27) whereas

Fig. 7.6.2 represents the Weblogo (generated from: http://weblogo.berkeley.edu/logo.cgi)

[Schneider et. aI., 1990; Crooks et. at., 2004] of P28 proteins. As represented in Fig.

7.6.2, the twenty cysteine residues are totally conserved along with other residues. These

conserved cysteines provide structural scaffold to Pb28 protein forming ten disulfide

bonds and this scaffold is conserved throughout the P28 family. Also residues that make

contacts among domains 1, 3 and 4 of Pb28 protein to form triangular shape are

conserved as shown in Table 7.5.1. Due to the presence of so many conserved cysteines,

it is predicted that proteins of P25 and P28 families evolved as a result of gene

duplication and hence should contain similar folds and domains. This prediction is further

supported by the results we obtained in this study.

80

The Pb28-13.1 Antigen-Antibody Complex The Pb28 model from P28 family contains four EGF domains arranged in

triangular faschion similar to Pvs25 protein as shown in Fig. 7.6.3(A). EGF-domains

have high tolerance to polymorphism and mutations and are tolerant to insertions and

deletions, therefore a high level of similarity between the template and query protein was

expected. The structure of Pb28 protein was predicted similar to the template protein

Pvs25 [Saxena et. al., 2004; Saxena et. ai., 2004; Saxena et. at., 2006] because of the

presence of 20 conserved cysteines, 37% sequence identity and 52% sequence similarity

(residues taken as similar: GA VLI, FYW, CM, ST, KRH, DENQ, P) between the

template protein Pvs25 and target protein Pb28.

7.3.1 Template

The mature sequence of Pb28 protein from Plasmodium was taken from Swissprot

database (Swiss-Prot id=Q04620). The sequence ofPb28 protein is as follows:

>Q0462010S24_PLABA 24 kDa ookinete surface protein - P~asmodium

berghei (strain Anka) .

MNFKYSFIFLFFIQLAIRYNNAKITVDTICKGGKLIQMSNHYECKCPSGYALKTENTCEPIVKCD

KLENINKVCGEYSICINQGNFGLEKAFVCMCTNGYMLSQNICKPTRCYNYECNAGKCILDSINPN

NPVCSCDIGKILQNGKCTGTGETKCLLKCKAAEECKLTGKHYECVSKPQAPGTGSETPSNSSFMN

GMSIISIIALLVIYVIVM

Only the mature protein was modeled i.e. MNFKYSFIFLFFIQLAIRYNNA

sequence was removed from the C-terminal and GMSIISIIALL VIYVIVM sequence was

removed from N-terminal before modeling of Pb28 protein. As already mentioned, the

cysteine arrangement of Pb28 is similar to Pvs25 (PDB code: lZ27) protein with

significant sequence identity of 52% (Fig. 7.6.1). Pvs25 protein was the only structure in

PDB database showing significant similarity to Pb28 protein and therefore Pvs25 was

used as template for modeling of Pb28 protein.

7.3.3 The Pb18 Model

The model of Pb28 protein indicates three EGF domains and fourth truncated EGF

domain arranged in the form of a triangular prism as represented in Fig. 7.6.3(A).

Structural superimposition of Pb28 model with template Pvs25 shows similarity in the

over all fold of the two proteins. As compared to the template protein Pvs25, Pb28

81

The Pb28-J3.1 Antigen-Antibody Complex protein contains maximum variations in the Bloop ofEGF domain II and in EGF domain

III as represented in the Fig. 7.6.3(B). A comparative study of the Pb28 protein with

Pvs25 protein showed that out of the four EGF-domains of Pb28, EGF domain I and N

have 4 cysteines. EGF domain I of Pb28 shows maximum similarity to EGF domain I of

Pvs25 with average RMSD of 1.21 A and maximum RMSD of 2.24 A whereas EGF

domain III of Pb28 shows maximum deviation to EGF domain III of Pvs25 with

maximum RMSD of 5.58 A and average RMSD of 2.79 A. EGF domain II has a

maximum RMSD of 5.07 A whereas average RMSD for EGF domain II is 2.54 A. In

case of EGF domain IV, average RMSD is 3.26A. In Pb28 EGF domain IV is larger in

comparison to Pvs28 and therefore calculating maximum RMSD was not possible in this

case. Ramachandran plot statistics of the Pb28 protein models showed that only one

residue Asn96 was present in the disallowed region as shown in Fig. 7.6.4. This Asn96

residue is present just before the disulphide bonded cysteine and change in its

conformation leads to breakage of the disulphide bond and therefore it was decided to

keep this residue as it is and no rotamers were searched. Prosa Web analysis revealed that

the Pb28 model falls within the permitted regions with a Z-score of -5.42 as shown in

Fig. 7.6.5(A) and the model obtained was found energetically favorable as represented by

the energy plot shown in Fig. 7.6.5(B).

7.3.4 Electrostatic representation Pbs28

The two faces of the Pb28 molecule reveal most of the positively charged residues on the

surface as shown in Fig. 7.6.6. In contrast to the other members of the P28 family (we

have studied six members of the family), this molecule reveals an overall positively

charged surface with a total charge of +3.00 kT. The positively charged EGF domain II

as shown in Fig. 7.6.6 (A) interacts with the negatively charged patch present on the scFv

of 13.1 antibody (Fig. 7.6.10). Edge II was found significantly positively charged on the

dorsal surface as shown in the Fig. 7.6.6(A). On ventral surface, EGF domain III showed

maximum positive residues on the surface. Negatively charged residues were few and

were concentrated primarily near the N-terminal and C-terminal pores of the molecule.

Pb28 is positively charged whereas the template Pvs25 is negatively charged (Total

charge= -10.00 kT). There is a minimal hydrophobic core with comparatively few

82

The Pb28-13.1 Antigen-Antibody Complex residues that are buried; most of the residues are solvent-accessible. The positively

charged residues are mainly situated on the surface of the molecule with relatively high

accessible molecular surface indicating the probable significance of these residues in

parasite's interaction to the host cell receptors. Cysteines are present in the core region of

the Pb28 molecule with least relative surface exposure which explains why cysteines are

conserved in all the members of the P28 family of proteins. In order to identify the

residues important in interaction of ookinete to the midgut cells of mosquito, we

compared the theoretical model of Pb28 with other functionally similar proteins of the

P28 family. A similar cysteine arrangement and high sequence and structural identity

between the members have been observed. Though members of P28 family have similar

functions and all of them are supposed to have four EGF domains, they differ in their

loop regions of the EGF domains and probably that is why they are able to recognize

different antibodies and molecules. It was striking to fmd that inspite of so much

similarity in P28 sequences; the charge distribution on the surface of the molecules

differs significantly. Differential charge distribution on the surface of P28 proteins

indicates towards different functional requirements of these molecules inside the

mosquito midgut.

7.3.5 The 13.1 Antibody Model

The model of scFv of 13.1 antibody was developed with the help of WAM server

[Whitelegg et. at., 2000]. Model was further refined with the help of Modeller and

ModLoop programs as shown in Fig. 7.6.7(A and B). The refinement led to major

changes in loop conformations as indicated by the Fig. 7.6.7(C). This antibody has been

reported as an efficient transmission blocking antibody against Pb28 protein [Yoshida et.

aI., 1999; Yoshida et. aI., 2001]. The final model obtained was evaluated with the help of

Procheck, WhatIF and ProsaWeb. Ramachandran plot statistics of the antibody model

showed that no residue was present in the disallowed regions as shown in Fig. 7.6.8.

ProsaWeb analysis revealed that the Z-score (-6.39) of scFv of the 13.1 model falls

within the permitted regions as shown in Fig. 7.6.9(A) and the model obtained is

energetically favorable as shown in Fig. 7.6.9(B). To show the charge distribution on the

surface of the antibody, electrostatic presentation of the molecule was made with the help

83

The Pb28-13.1 Antigen-Antibody Complex of GRASP [Nicholls et. al., 1991] as shown in Fig. 7.6.10. The antibody clearly

represents negatively charged surface (Total charge = -5.0 kT) in contrast to the

positively charged Pb28 protein.

7.3.6 Docking studies

To gain more understanding about transmission blocking mechanism, we docked the

models of Pb28 protein and scFv of 13.1 antibody with the help of ZDOCK program and

obtained models were refined and reranked with the help of RDOCK program. The top

30 complexes obtained were screened according to the biochemical studies available

[Yoshida et. ai., 1999; Yoshida et. ai., 2001]. All the complexes interacting with EGF

domain II were taken and checked for antigen-antibody type of interaction. The

complexes not interacting with CDRs of the scFv of antibody 13.1 were removed. The

complexes representing interactions with CDRs were selected and the best probable

complex was then chosen on the basis of maximum buried surface area. It was found that

EGF domain II of Pb28 interacts with both light and heavy chains of scFv of 13.1

antibody forming two hydrogen bonds each (Fig. 7.6.11). The heavy chain forms 80,

whereas light chain forms 136 non-bonded contacts along with the hydrogen bonds (Fig.

7.6.12). The buried surface area between Pb28 protein and scFv of 13.1 antibody was

calculated to 1940.3 A2. Fig. 7.6.12 represents the detailed interactions at molecular level.

The figure was created with the help ofLIGPLOT software [Wallace et. at., 1995]. In the

complex of Pvs25 protein with scFv of 2A8 antibody, only light chain of the antibody

participates in the complex formation, whereas in case of Pb28 protein and 13.1 antibody

both light and heavy chains of the scFv equally participate in complex formation. The

study clearly demonstrates that variability in loop regions leads to specificity for the

monoclonal antibodies and therefore each P25 and P28 molecule recognizes its specific

monoclonal antibody.

7.4 Conclusion In this study, we have carried out a detailed bioinformatics analysis of the sequence and

structure of Pb28 in order to gain understanding of the functional aspects of this protein.

Comparative studies between theoretical model of Pb28 (Modelled by us) and

84

The Pb28-13.1 Antigen-Antibody Complex functionally similar Pvs25, indicated that EGF domain I of Pb28 shows maximum

similarity to EGF domain I of Pvs25 whereas EGF domain III showed maXlmum

deviation. Maximum variations were found in the loop regions. These loop regions are

responsible for variable molecular recognition among P25 and P28 proteins. These

studies also indicate towards the probable differences in the active sites of these proteins

and may help in explaining why EGF domain II of Pb28 interacts with scFv of 13.1

antibody. Antigen-antibody interaction study of Pb28 and scFv of 13.1 antibody showed

that EGF domain II of Pb28 interacted with both light and heavy chains of scFv of the

transmission blocking antibody 13.1 (Fig. 7.6.11) forming two hydrogen bonds each.

Buried surface area between the two was calculated to be 1940.3 A2. Our study has

drawn attention to the functionally important region and provides a pointer to

experimental work to validate the observations made here based on in silica analysis. It

has been shown earlier by Spano et. al. (1996) [Spano et. al., 1996] that the Gly-Leu-Glu­

Lys-Ala-Phe-Val-Cys sequence on the B loop of EGF Domain II of Pb28 interacts with

13.1 antibody which provides an experimental support to our study .. The study also

demonstrates that the interaction of the ookinete surface proteins to their respective

transmission blocking antibody is likely to differ in P28 proteins and that loops are the

major areas, where the specificity of transmission blocking antigen-antibody interaction

lies.

85

The Pb28-13.1 Antigen-Antibody Complex

7.5 Tables

Table 7.5.1: Conserved domain interactions between Pb28 and Pvs25

(Note: In the column Atom name, E represents epsilon, G represents gamma and Z represents zeta.)

<-----A TOM 1-----> EGF- <-----A TOM 2 ----> EGF- H-Bond Atom I Atom I Res I Res domain Atom I Atom I Res I Res domain Distance no. name name no. involved no. name name no. involved 109 NE2 GLN 15 I <--> 997 0 LYS 132 III 2.88 120 N SER 17 I <--> 869 0 CYS 114 III 2.68 123 OG SER 17 I <--> 864 N CYS 114 III 2.66 126 N ASN 18 I <--> 861 OG SER 113 III 2.96 136 N01 HIS 19 I <--> 861 OG SER 113 III 2.73

36 001 ASP 5 I <-->1026 NZ LYS 136 IV 2.70 103 N GLN 15 I <-->1136 0 TYR 150 IV 3.08 111 0 GLN 15 I <-->1125 N TYR 150 IV 2.95 108 OE1 GLN 15 I <-->1004 N LEU 134 IV 2.88 220 N LEU 30 I <--> 409 OE1 GLU 54 II 3.06 227 0 LEU 30 I <--> 413 N TYR 55 II 2.71

Table 7.5.2: Pbs21 and TBmAb 13.1 interaction interface statistics

(Note: In the column Atom name, E represents epsilon, G represents gamma and Z represents zeta.)

Chain No. of Interface No. of No. of No. of No. of Interface area (A2) salt disulphide Hydrogen Non-bonded residues bridges bonds bonds contacts

A-H 8:11 386:368 - - 2 80 A-L 15:14 665:718 - - 2 136

Hydrogen bond formation between Pb28 protein and scFv TBmAb 13.1

S. <-----A TOM 1 -----> <----- A TOM 2 ----->

No. Atom I Atom I Res. I Res. no. Atom I Atom I Res. I Res. no. I Bond no. name name & Chain no. name name & Chain length

Hydrogen bonds formed by light chain

1 2737 10 I ASN I 288(A) 491 I OG I SER I 56(L) 12.72),. 2 2928 10 I SER I 309(A) 704 I N I GLU I 81(L) 12.67 A Hydrogen bonds formed by heavy chain

1 2745 INE2 I GLN I 289(A) 2074 10 I ASP I 217(H) I 2.73 A 2 2815 IN I ALA I 297(A) 2079 ISG I CYS I 218(H) 12.82A

86

7.6 FIGURES A 10 20 30 '90 50

Pb28 Pvs25

Pb28 Pvs25

Pb28 Pvs25

Pb28 Pvs25

B

r ~ ~

~ .. c: Q)

::E

... ~~~.~.:iI 1 2 3 4 5

60 70 80 90 100

.. I .. . I .... ;,'b~i<j:\~4tG~~N&~~~·, . "&~4i Pf\QVNMYftI4IJrSKlm~LD'I;2 KlJ;E$

6 7 8 9 10 11 12 110 140 150

iiLD~IN~~~~' IlVX·,'LSEIQS

13 1415 16 17 18 19 160 170

LTGK SKPC!l\rCIrGSXTP.'IlSSFMI'~ •••••••• 1 •••• 1 •••• ' •••• , ••••

rNEG ;2C---MElJFTFDKEIflvCLGP

20

100 I I I I I ~

90 80 70

r----% Identity=36.9% ~

~ ~-:~ Crit. % Identity= 42% =- .--

60 50 40

-. - --. 1 . /\ -. " .- {,.liJ -.. - 1 1 .- -~ ''/'' r' I, -- -

30 20

r--. "\1\ n._ .... r.J' .. \,...J~, / I~I

\jl'\r"

.

r- J L' 10 l-

0 I I I I I I I I

20 40 60 80 100 120 140 160 180

Alignment position -----+.

Figure 7.6.1: (A) Sequence alignment of query sequence Pb28 belonging to P28 family of ookinete surface antigens with template sequence Pvs25 (PDB id lz27). Completely conserved residues between the two sequences are shown in black background whereas the semi conserved residues are shown in gray background. The 20 conserved cysteines are indicated by brick red vertical rectanglcs in the background. Negatively charged (D and E), positively charged (K and R), polar (N, Q, S and T), aliphatic (I, L, M and V) and aromatic (F and Y) residues are shown in red, blue, bluish-purple, green and cyan color respectively. Histidine, glycine, proline are represented in pink, buff and gray color respectively. (B) represents the numerical and graphical form of the percent identity between target (Pys25 starting from AITP A) and template (Pvs25) sequences.

Weblogo: P28 family

EGF domain I

4

3 EGF domain II

~2D' _x~~~Jl{~~fl~ ~ ~e~~ ~gL~~~ N-NM.~~~G~~=~~~~~~S~~N~~~~~~~~~M~R~~~~~~~·~~~~~~~c

4

4

3

EGF domain III

EGF domain IV

Figure 7.6.2: Weblogo representation for 6 sequences of P28 family of ookinete surface antigens. All the members are represented in Table-7.S.1. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid. Negatively charged (D and E), positively charged (K and R), and cysteine (C) residues are shown in red, blue and yellow color respectively whereas rest all residues are in black color. Conservation of cysteine residues in all the four EGF domains is well represented by the logos above.

A

B

EGF domain I EGF domain II

• EGF domain III • EGF domain IV

Figure 7.6.3: A) Cartoon representation of the model of Pb28. The model was generated assuming that the disulfide connectivity of 20 cysteines (shown in yellow CPK representation) in Pb28 is the same as that in the template Pvs25 (PDB id 1Z27). Prs25 model shows four EGF domains as shown above. All the four EGF domains of the molecule are shown in different colors with EGF-domain I in blue, EGF-domain II in red, EGF-domain III in gray and EGF-domain IV in orange color. B) Superimposed structures of Pb28 and Pvs25(C-alpha atoms only) after structural alignment using program STAMP. Superimposition between theoretical 3D model of Pb28 and template Pvs25 shows similarity in the overall fold of the two proteins. EGF domain III and B-Ioop ofEGF domain II shows maximum variations.

180

135

90

45 --- a Vl

<!.) <!.) • :... bJ)

0 <!.) "0 '-" Vl

0...

-45

-90

-135 b

I b

·-1

. J

• ...!.. . ~ • •

•

•

f-l l - p .. • I

Phi (degrees)

Plot statistics

Re,idues inlllost favolU'ed regions [A-B.L) Re,idues in addition.1l allo\yed regions [a.b.l.p) Residues in generously a]]O\yed regiom [-a.-b,-l.-p] Residues ill dis.111owed regions

Kumber of non-glycine ond non-proline residues

Kumb.:r of end-residues (excl. Gly and Pro)

Kumb.:r of glycine residues (shown as triangles) Kumber of proline re.,idues

T otaillumber of residues

. ~

ASN ~6 (G)

'"

122 24 1 1

148

.:!

15 8

173

- b '" b

CYSJJ2 (~ •

'"

'"

- b

82.4% 16.2% 0.7% 0.7%

100.0%

Figure 7.6.4: Ramachandran plot for theoretical 3D model of Pb28 using x-ray crystallographic structure of native Pvs25 protein (PDB id lZ27) as a template. Number of residues in most favored regions were 82.4%. Number of residues in allowed region 16.2%. Residues in generously allowed regions 0.7% and number of residues lying in outlier region were 0.7 %.

A

Z-score=-S.42

-15

3r-------------------~~~~~

B 2

1

.3~,------------------.,..'~7 Sequence position

Figure 7.6.5: ProsaWeb analysis of theoretical 3D model ofPb28. (A) represents the Zscore plot of the Pb28 model where all the values lie within the normal range whereas (B) represents the energy plot for Pb28 model. Energy for all the residues is below zero or negative indicating the stability of the molecule.

Figure 7.6.6: Electrostatic representation of theoretical structural model of malaria transmission blocking surface antigen Pb28 prepared with the help of GRASP. Surface potential was taken from -lOkT (red) to +lOkT (blue). Figure (A) represents the front view of Pb28, (B) the opposite face after 1800 rotation, (C-E) view of each edge of the molecule. ProsaWeb, PROCHECK and WhatIF programs were used for statistical evaluation of the final model. As represented by the different views of the molecule, in contrast to other members of P28 family the negatively charged residues are very few and the molecule is positively charged overall. EGF domain II is more positively charged (Total charge = +3 .0) on the dorsal side whereas EGF domain III is more positively charges on the ventral side of the molecule represented in blue color. The orientation in Fig (B) is the same as in Fig 3(A&B).

A B

c

Figure 7.6.7: A) Cartoon representation of the model of scFv region of transmission blocking antibody 13 .1. The model was generated with the help of web antibody modeling (W AM) server. B) Final model obtained after refIning scFv of 13.1 (W AM model) with the help of Modeller and ModLoop. C) Superimposed W AM model and fInal model of scFv of 13.1 to show the changes occurred after refInement of the model.

180

135

90 •

45 ~ ' a IJl V V :.... 01)

0 v "0 '-" IJl

0... .!.... -45

-90

-135 -I b

·-1

.I · 1

... ~ ~

4

•

~ 4

I L1RJ 50 (A) I • · .p · • I

: ~ 4

5 Phi (degrees)

Plot stntistics

Residuo:~ in most favolU·ed region; [A.B.L] Re~idue~ in additional allowed regions [a.b.l.p 1 Residue~ in generously allowo:d regions [-a. - b. - I. -p 1 Residue; in di>'111o\\'ed region;

Kumber of non-glycine and lion-proline ro:sidues

Klllllbet· of end-residue, (excl. Gly and Pm)

KlUllbet· of glycine residue; (;hown as trianglo:s) "lUuber of proline residuo:;

T otalmuuber of ro:sidues

162 28 1 0

191

4

22 11

228

~b .4 b

4

4

4 - b

84.8% 1.:1.7% 0.5% 0.0%

100.0%

Figure 7.6.8: Ramachandran plot for theoretical 3D model of transmission blocking monoclonal antibody (TBmAb) 13.1 using X-ray crystallographic structure of native Pvs25 (PDB code lZ27) as a template. Number of residues in most favored region (84.8%). Number of residues in allowed region (14.7%). Residues in generously allowed regions = 0.5%. Number of residues lying in outlier region = 0.0 %.

A

B

~ o o I/)

N

Z-score=-6.39

Number of residues ----+.

t,

,'~ ________________________________ ~~ Sequence position ---..... -

Figure 7.6.9: ProsaWeb analysis of theoretical 3D model of scFv of 13.1 antibody. (A) represents the Zscore plot of the 13.1 model where all the values lie within the normal range whereas (B) represents the energy plot for scFv of 13.1 antibody model. Energy for all the residues is below zero or negative, indicating the stability of the molecule.

Figure 7.6.10: Electrostatic representation of theoretical structural model of malaria transmission blocking antibody 13.1 prepared with the help of GRASP. Surface potential was taken from -10kT (red) to +1 OkT (blue). Figure (A) represents the front view of Pb28, (B) the opposite face after 1800 rotation, (C-E) view of each edge of the molecule. Prosa Web, Procheck and WhatIF programs were used for statistical evaluation of the final model. As represented by the different views of the molecule, the negatively charged residues are present on the junction of the heavy and light chain and more towards the light chain represented in red color. Total charge on the surface ofthe molecule = -5 .00.

A

B

Pb28 Model Heavy chain Light chain

Pb28 Model Heavy chain Light chain BLoop EGF domain II

-

--

Figure 7.6.11: Figure represents the model of scFv region of transmission blocking antibody 13.1 (light chain shown in yellow and heavy chain shown in magenta color) and the interaction of this region with the Pb28 model shown in cyan color. The EGF domain II of Pb28 interacts with light and heavy chain of scFv 13.1 forming two hydrogen bonds each. The light chain makes 136 non-bonded contacts whereas heavy chain forms 80 non-bonded contacts. A) scFv of TBmAb 13.1 shown in licorice presentation and Pb28 model shown in surface presentation B) CDRs of TBmAb 13.1 shown in surface representation whereas Pb28 model and rest scFv shown in cartoon representation. B loop of EGF domain II which interacts with TBmAb 13.1 is shown in dark blue cartoon representation.

A)

1111111 Non bonded contacts

- Hydrogen bonds

B

Light chain

Chaiu A Chain L

Gly290 ,)),~ I, IHII_ Glu35 " I .... /f/

Asn188 Se1'56

De287 "

Glu289

\",,1299

l\Iet301

C~'S3000 Gln310 _ ... " ,l l1 l1 '·

.,.i{!~/ l:

Ll'u308

Sl'r309

l\Il't307

T)T306

Cys302 0 ' " . .1. .lt

t'

Tyr319 1111 1111

\",,158

Gly57

Arg61

Glu80

Glu81

Glu79

Ala60

Pro39

Yal78

Pro77

Gly13

Ll'u14

c

Heavy chain

Chain A

Figure 7.6.12: A) Schematic representation of the A chain (Pb28) protein interaction with heavy and light chains of scFv of 13 ,I antibody. B) Residue interaction details of Pb28 and light chain of scFv 13.1 antibody. C) Residue interaction details of heavy chain of scFv 13.1 with Pb28 (A-chain). Non bonded interactions are shown in vertical orange lines whereas hydrogen bonds are shown by blue lines connecting the residues.