Structure q[Pka-DBL - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/16834/9/09... · 2018-07-09 · Structure of Pka-DBL 2.0 Introduction Malaria pathogenesis is mediated by

Structure q[Pka-DBL

Structure of Pka-DBL

2.0 Introduction

Malaria pathogenesis is mediated by erythrocyte invasion and cytoadherence,

where Plasmodium specific modules called Duffy-binding like domains (DBLs) play an

instrumental role (Miller et al. 2002). These cysteine-rich domains recognize and bind to

a variety of host cell surface receptors on erythrocytes for invasion, and on host

endothelium for cytoadherpnce. Since DBLs from parasite EBPs are involved in

merozoite invasion and PfEMP1 DBL domains are implicated in cytoadherence, they

present themselves as strong candidates for vaccine/ drug development.

The simian malaria parasite P. knowlesi and the human parasite P. vivax both use

Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes

(Miller et al. 1975). The DBL domains from P. vivax and P. knowlesi EBPs, Pv-DBL and

Pka-DBL, are 71% identical in sequence. These are homologous to the two tandem DBL

domains found in P. falciparum EBP (EBA-175), which invades human erythrocytes

through its interaction with sialic acid residues on glycophorin A (Miller et al. 2002). A

sulfated tyrosine (Tyr41) in human DARC is a key component of the Pv/Pka-DBL:

DARC interaction (Choe et al. 2005), and this dependence is similar to the role of a

sulfated tyrosine in the gp120:CCR5 interaction during HIV invasion (Farzan et al.

1999).

The complete dependence of P. vivax on EBP: DARC interaction for the crucial

step of junction formation during invasion surfaces as an opportunity for development of

an effective intervention strategy. This work focuses on structure solution of Pka-DBL

domain to angstrom resolution using X-ray crystallography, and a comprehensive

analysis of its interaction with human DARC.

2.1 Materials and methods

2.1.1 Production of recombinant P. knowlesi Duffy-binding domain (Pka.-DBL)

Pka-DBL was expressed in E. coli, purified from inclusion bodies and refolded

into its native conformation using methods similar to those described previously for the

production of a recombinant Duffy-binding domain from P. vivax (Singh et al. 2001).

DNA fragments encoding the binding domain of P. knowlesi Duffy-binding protein

(amino acid residues 200-536) fused to a C-terminal hexa-histidine tag were PCR

32


amplified using gene specific primers and plasmid pGP67BKADR2.1 (Singh et al. 2002)

encoding Pka.-DBL as a template. The PCR product was digested with Neal and Notl

restriction enzymes and cloned into expression vector pET28a (Novagen, Darmstadt,

Germany) to yield plasmid pET28a-pkadbl. Expression of Pka.-DBL was carried out in

E. coli B834 cells by growing transformed cells to an OD6oo of 0.6-0.8, followed by 0

induction with 1 mM·IPTG for 3-4 hat 37 C. Following induction, the cells were lysed

by sonication and.inclusion bodies were collected by centrifugation at 12 000 g for 45

min, which were solubilized in 6 M guanidinium chloride. Solubilized inclusion bodies

were subjected to Ni-NTA affinity chromatography to purify Pka.-DBL using a pH

gradient. Recombinant Pka.-DBL was refolded by the method of rapid dilution, as

described before (Singh et al. 2001). Briefly, purified recombinant Pka.-DBL was diluted

-50-fold in refolding buffer containing 50 mM phosphate buffer (pH 7.2), 1 mM GSH,

0.1 mM GSSG, 1 M urea and 0.5M arginine. The final protein concentration while

refolding was maintained at a maximum of 33mgll. Refolding was allowed to proceed for 0

36 h at 10 C. After removal of arginine by dialysis, refolded Pka.-DBL was purified by

ion-exchange chromatography on a SP-Sepharose FF column (Amersham Biosciences,

Bucks, U.K.). The protein was eluted from the SP-Sepharose FF column using a salt

gradient and was purified further to homogeneity by gel-exclusion chromatography using

a Superdex 75 column (Amersham Biosciences).

2.1.2 Protein crystallization and data collection

Purified Pka.-DBL (15 mg/ml) was crystallized in O.lM HEPES pH 7.5, 12%

polyethylene glycol 10,000, 0.1M ammonium sulphate and 0.5% N-octyl ~-D

glucopyranoside. The crystal used for single-wavelength anomalous dispersion (SAD)

data collection was grown in 10 mM potassium aurocyanide with 2% v/v dioxane, 0.1M

bicine pH 9.0 and 10% polyethylene glycol 20,000. All crystal forms belong to the space

group P43212 with a= 60.57 A0, b = 60.57 A0

, c = 241.04 A0 and with one monomer per

asymmetric unit. Crystals were soaked in mother liquor supplemented with 20% ethylene

glycol for 30 s and then flash-frozen before data collection. The native and heavy atom

co-crystals were extremely sensitive to both temperature changes and X-rays and

33


diffracted poorly at in-house sources. All diffraction data were collected using

synchrotron radiation under cryogenic conditions at the BM14 beamline, ESRF,

Grenoble. Relevant data collection and refinement statistics are provided in Table 2.1.

2.1.3 Structure determination and analysis

Diffraction data were collected on a 50 x 50 x 100 J..Lm protein crystal of Pka

DBL co-crystallized with gold. The Au-absorption edge was determined by a

fluorescence scan and 360° of data were collected in 1.0° oscillation frames using a

MARCCD detector (Table 2.1). Native diffraction data to 3.0 A0 were also collected at

the BM14 beamline. The data were indexed and scaled with DENZO and SCALEPACK

programs (Otwinowski and W. 1997). Processing of the SAD data set was done using

SHARP (de La Fortelle and Bricogne' 1997) and SOLVE (Terwilliger and Berendzen

1999), and each program identified two gold sites. SAD phases, after density

modification (solvent content of the crystals is 62% ), were used to calculate a native map

to 3.3A 0 , which was of moderate quality although the secondary structure elements

(helices) were clearly visible. Backbone and side-chain tracing of the protein structure

was done using the program 0 (Jones et al. 1991). Crystallographic model refinement

was initiated using SAD data in CNS (Brunger et al. 1998), and the model was

subsequently refined against native data to 3 A 0 • A total of 6% (or 556) of the reflections

were kept aside for Rfree evaluation before start of the refinement. The final model

statistics are given in Table 2.2; there are no residues in disallowed regions of the

Ramachandaran plot and the model stereochemistry is good, as verified by PROCHECK

(Laskowski et al. 1993). Where side chain positions are not clear, some residues in the

final model have been modelled as alanines. ln addition, no reliable model could be built

for residues 1-14, 53-63, 181-185, 263-268 and 308-337. Structural superpositions

between F1/F2 (PDB accession code 1ZRL) and Pka-DBL were done using LSQMAN

(Kleywegt 1999), and the residue limits along with root mean square deviations are F1 (8-

282)- Pka-DBL(15-307), 1.6 A0 (for 195 Ca atoms); F1(8-282)- F2 (297-596), 1.9A0

(for 185 Ca atoms), and F2(297-596)- Pka-DBL(15-307), 1.8A0 (for 156 Ca atoms). " All figures were made using BOBSCRIPT (Esnouf 1997), RASTER3D (Merritt and

Bacon 1997) or GRASP (Nicholls et al. 1991).

34


2.1.4 Binding of Pka-DBL to recombinant sulfated DARC

Binding specificity of recombinant Pka-DBL to human DARC was determined

using ELISA-based assays according to published protocols (Hans et al. 2005). Briefly,

recombinant nDARC, YF-nDARC (a mutant version of human DARC where the active

tyrosine 41 was altered to phenylalanine) and BSA were coated in sodium bicarbonate

buffer pH 9.6 overnight ,at 4°C at a concentration of lllg/ml in ELISA plate wells.

Recombinant DBLs (0.05 llg/ml) were added to the wells to allow binding. Bound

proteins were detected with rabbit/mouse protein specific antisera and anti-rabbit or anti

mouse IgG horseradish peroxidase-conjugated secondary antibodies. Experiments were

done in triplicates, using appropriate controls.

Wavelength (A) • Range: (A)

Reflections Rmerge (%)t Completeness (%)

Native

0.88560 20-3.0 9,599

7.5 (483) 98.7 (97.6)

kKeeping Bijovoet pairs separate during data p;rocesslng.

Gold·SAD

103927 50-.3.3 7}600*

9.5 (372) 100 (100)

tR merse = I:( I - < I> )IE < I >; numbers in parentheses correspond to values in the highest resoiLition shelL

Table 2.1: Diffraction data collection and model refinement statistics.

Resolution (A) R wed:! R tree~ Number o·f atoms

Protein ligand/ion Water

S-f actors Protein Uga11d/fon Water

r.m.s deviations Bond length <A> Bond angles (0

)

20.0-3.0 24.8/31.2

2,186 0

42

71 0 43

0.014 1.9

·Rwark =E[(IFobsl)- (IFesl(:ln/ECIFm~· R1~ is the same as RwM. but comprises a 'test' set of reflections (fraction 6%; number 556) that were excluded during model refinement""'.

Table 2.2: X-ray refinement statistics.

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2.2 Results

2.2.1 Expression and purification of PkaDBL in a soluble and functional form

The recombinant P. knowlesi Duffy-binding domain expressed in E. coli

accumulated in inclusion bodies. The protein was extracted and refolded into its

functional form, yielding -1 mg/1 of refolded and purified protein from shake flask

cultures. As expected, this protein migrates on SDS/PAGE as a single band of approx . 40

kDa (Figure 2.1 a). The recombinant P. knowlesi Duffy-binding domain is monomeric,

based on its migration in gel-filtration columns (Figure 2.1 b).

6 90 .00

E s9o.oo t:

0 49 0 .00 QC) N - 390.00 C) u 16 290 .00

..c 0 190.00 II)

..c ~ 90.00

·10 .00 ~--......----...or----...::::r-----.---0 .00 5 .00 10 .00 1 5.00 20 .00 25 .00

Volume(ml)

Figure 2.1: Recombinant Pka-DBL. a: SDS-PAGE analysis of purified

PkaDBL b: Gel permeation chromatography profile of Pka-DBL on S75 superdex

column

2.2.2 Protein crystallization and data collection

We determined the crystal structure of recombinant Pka-DBL (337 residues) by

collecting single-wavelength anomalous dispersion data from a crystal of Pka-DBL

soaked in potassium gold cyanide (Table 2.1 ). Protein crystals of Pka-DBL could only be

obtained in the presence of detergent N-octyl ~-D-glucopyranoside (Figure 2.2). The

initial electron density map was calculated to 3.3A, and data from a nati ve crystal that

diffracted better was used to refine the model to 3A.

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Figure 2.2: Pka-DBL crystal

2.2.3 Structure of Pka-DBL

The structure of Pka.-DBL represents an all-helical, boomerang-shaped,

spherically curved, monomeric assembly of three distinct subdomains (Figure 2.3). Its

overall structure is similar to F1/F2 DBLs of EBA-175 (Tolia et al. 2005). The molecule

comprises of twelve a.-helices and six invariant disulphide bridges. Residues 15-52

constitute a random-coiled subdomain 1, while residues 64-180 comprising helices a.1-

a.6 make up subdomain 2. A short linker segment connects subdomain 2 to subdomain 3,

where the latter extends from residues 186-307 encompassing helices a.7-a.12. The

subdomains 1, 2 and 3 contain two, one and three disulfide bridges respectively, which

result from the twelve invariant cysteine residues that are characteristic of all DBLs

(Figure 2.4 a, b and c). The segment that links subdomains 1 and 2 (residues 53-63) is

missing and likely to be flexible. This is consistent with data from our earlier proteolysis

experiments, which had shown the presence of this disordered linker, beginning from

residue Arg57 (Singh et al. 2003). The interior of subdomain 2 is comprised of a

hydrophobic core that runs along the length of a.l. Subdomain 3 is built of two -40 A long helical towers ( a.7 and a.9) that are connected by a solvent exposed linker, termed

subdomain 3 loop that rests on top of helices a.7 and a.9. The segment is bound to

subdomain 3 and has three neighbouring disulfide bridges that hold it in place. A less

pronounced hydrophobic core within subdomain 3 establishes links between the rest of

a.7 and a.9. Interestingly, subdomain 1 nestles equally between subdomains 2 and 3 and

makes hydrophobic interactions with them. Any inter-subdomain disulphide bonds are

37


absent from Pka-DBL, and the three subdomains remain connected to each other either

by means of hydrophobic interactions or by flexible linkers.

a

b

Figure 2.3: Orthogonal views of Pka-DBL.

Domain 1 -Pk-EBP

Pv-EBP

run Pf-EBA175 ;::::;===~::::::;==::::=~===--~ ~-~ Pf-EMP1

Figure 2.4 a, b: Disulphide linkages in Pka-DBL

38

Pk Pv PfFl PfF2 Var-g Var-a

Pk Pv PfFl PfF2 Var-g Var - a



Pk Pv PfFl PfF2 Var-g Va r-a

1 1


NDK-RKRGEROWD PAEKD-----I YK-RKRRERDWD NTKKD-----V

1 -MRINNGRNTSSNNEVLSN REK-RK--G KKK~-SNY

1 KNSVDTNTKVWE KNPYI(!ffiKD 1 HPKKNSNGYPDWQ GNINLVEDPRV 1 MEYTEGRKPCYERNEKRFSNEGEAKCGSDKIRDYGIKSAG---GA

<11

46 MKGLTNLVNNTRTHSHN-------DITFLKLNLKRKLMYDAAVEGDLLLK----K 46 MKELTNLVNNTDTNFHR-------DITFRKLYLKRKLIYDAAVEGDLLLK----L 51 IVNLSIIK---- - -------------TYTKETMKDHFIEASKKESQLLLK-- --K 53 LGNIDRIYDKN------------------LLMIKEHILAIAIYESRILKR----K 50 VHFLANDNEIKKLQSQVNLKEAFIKSAAAETFFSWYY\'KSKDGEGNELDK---EL 53 DRNLEYLINKN---------------TNTTHDLLGNVLVTAKYEGDSIVNNHPDK

90 90 84 86

"-"'"-.~K"'D""I""'R.~IGLGDFGDIIMGTNMEG-IGYSQVVENNLRQVFG ------

KDIR~ISLGDFGDIIMGTDMEG- IGYSKVVENNLRSIFG-- ----NDLKNSFLDYGHLAMGNDMDF-GGYS~KAEWK~KGAHGEI

KIINKTFADIRDIIGGTDYWN-DLSNRKLVGKINTN~---102 KEGKIPPAFLRSMFYTFGDYROFLFGTDISKGHGEGSKLKEQIDSLFKNGD----

93 NSSGNKSSI ALARSFADIGDIVRGRDMFK - PNDADKVEKGLQVVFGKIYNSLP

137 -----------TDEKAKQDRKQ~TWNESKEHIWRAMMFSIRSR LKEKF --------137 ----- - -----TDEKAQQRRKQWWNESKAQIWTAMMYSVKKRLKGNF--------137 -----------SEHKIKNFRKEWWNEFREKLWEAMLSEHKNNINN----------137 - ---- - ----- KNDKL~DEWWKVIKKDVWNVISWVFKDKT------------153 -----------QKSPNGKTRQEWWTEHSHEIWEAMLCALVKIGAKKDDFTENYG-147 SPAQKHYAHDDGSGNYYKLREDtTWAINRKEVWKAITCRAPNEAliFFRNISGNMKA

173 173 171 169

<17

196 --YNNVKFSDKSTTLEEFAKRPQFLRWLTEWYDDYCYTR KYLKDVQEK 202 FTSQGY GHSETNVPTNLDYVPQFLRt~DEWAEEFCRIRKIKLENVKKE

Pk 220 Pv 220 PfFl 213 PfF2 212 Var-g 249 Var-a 253


a.10 a.11

272 TAYDILKQELNGFKEATFENEINKRDN-LYNH 272 TPYDILKQELDEFNEVAFENEINKRDG-AYIELf V S 258 ----YLIKISENKNDAKVSLLLNNCDA-EYSK~ D-261 KPEVYLKKYSEKCSNLNFEDEFKEELHSDYKNKQ 286 ----- - KRFDRQHIGVMVTDYTGTNATDYLNR------286 ----- -----CENACSNYTKWIEIQRKQFDKQKRK---

Figure 2.4 c : Sequence alignm ent of homologous DBLs.

Shown are secondary structure elements for Pka-DBUPv-DBL (orange) , minimal

receptor-binding regions (yellow), conserved cysteine residues (green), proteolytic site

on Pka-DBL (a rrow), Pv-DBL clusters of polymorphic residues (grey) , mutations that

affect DARC recognition (red), contact residues between F 1-F2 and sialic acid (brown)

and Fl-F2 dimerization elements (black boxes).

39


Pka-DBL subdomain 3 is a prominent feature of this molecule with high

sequence conservation across DBL domains (Fig 2.4 c). The highly ordered subdomain 3

loop (Ala215 to Asn221) protrudes into the solvent emanating from the top of helices a7

and a9. Across Plasmodial species, both sequence and length of this loop varies

drastically (Fig 2.4 c). Multiple sequence alignment of DBL domains from EBAs reveals

the species specific nature of subdomain 3 loop (Figure 2.5) . Though the amino acid

sequence of this loop is identical in all polymorphic forms of PvDBL domains, it varies

among Pka- DBL and PvDBL domains despite their functional closeness (both these

DBL domains use DARC for invasion of human erythrocytes). The only conserved

residue in DBL domains of EBAs is Lys217 (Figure 2.5), which is fully solvent exposed.

K217 A 40 A r y

PkDBL 214 KLYYNNMAI 226 PvDBL 21 4 DGKI NYTDKK 226 PyDBL 21 5 TKKSNISTESI 22 7 PbDBL 226 TKKS IISTESI 238 PfF1 207 KNNTLY---E 2 1 6 PfF2 207 212 DBLy 2 43 251 DB La 200 RDEPNN---KY 209

Figure 2.5: Subdomain 3 loop. Solvent-exposed loop on top of helices a7

and o9 of subdomain 3 of the Pka- DBL domain. Multiple sequence alignment of

subdomain 3 loop from diverse DBL domains from EBPs and PfEMP-1 s ( PkDBL and

PvDBL, DBL domains from P. knowlesi and P. vivax Duffy binding proteins,

respectively; PyDBL and PbDBL, P. yoelii and P. berghei EBPs respectively; PJF1 and

PfF2 of P. falciparum EBA-175; DBLy, CSA binding DBL domain from FCR3var1 CSA;

DBLa, receptor binding domainfrom R29var). Positions of the conserved lysine (K217)

in the loop are shown.

40


2.2.4 Identification of DARC recognition site in Pka-DBL

The structural elements within Pv/Pka.-DBL that are required for binding human

RBCs were described earlier using a series of domain deletion and site-directed

mutagenesis experiments (Singh et al. 2003; VanBuskirk et al. 2004; Hans et al. 2005).

Based on these data, we have identified an invariant, surface exposed patch on

subdomain 2 which is likely to provide the contact residues Tyr94, Asn95, Lys96, Arg

103, Leu168 and Ile175 for DARC recognition on erythrocytes (Figure 2.6). Site-directed

mutagenesis of each of these residues significantly disrupts the Pv/Pka.-DBL: DARC

interaction (VanBuskirk et al. 2004; Hans et al. 2005). The nARC-recognition site is a

flat, dual-character surface composed of distinct hydrophobic (Tyr94, Leu168 and Ile175)

and hydrophilic (Asn95, Lys96 and Arg103) residues. On top of subdomain 2, the

hydrophobic channel constituted by Tyr94, Leu168 and Ile175 lies immediately adjacent

to a spread of positive charge density that includes Lys96, Lys 100, Argl03 and Lys177

(Figure 2.6 b, c). The twin-nature of contact surface described above mirrors well the

highly polarised character of sulfated tyrosines. Since sulfation of DARC Tyr41 is known

to increase Pv/Pka.-DBL: DARC affinity by up to a thousand fold, the dual-charge

character of DARC and its binding site are likely to be important for successful

interaction. Consistent with our description of the DARC interaction site, several single

site mutants that do not affect DARC binding fall outside the recognition surface. Other

mutants like Phe98 and Phe172, which alter DARC affinity for PvDBL are likely to have

purely structural roles because of their proximity to the binding site.

2.2.5 Binding specificity of Pka-DBL to DARC

Binding specificity of recombinant Pka.-DBL to human DARC was determined

using ELISA-based assays. Wild type DARC and a mutant version of human DARC

(where the active tyrosine 41 was altered to phenylalanine) were used in binding assays.

PvDBL and Pka.-DBL bound WT DARC strongly, while EBA-175 and AMA-1 did not.

On mutation of the critical binding residue Y41 on human DARC, no significant binding

to Pka.-DBL could be observed (Figure 2.7). Standard deviations in these experiments

were less than 10% of observed values.

41


c

Figure 2.6: DARC recognition site. a, Ribbon representation of

subdomain 2 (grey) with DARC binding residues shown in ball-and-stick representation.

The DARC-recognition site includes nonpolar (yellow) and polar residues (blue) . b,

Molecular surface representation of Pka-DBL showing the dual charge nature of DARC

contact residues. Subdomain I is shown in green, subdomain 2 in yellow and subdomain

3 in light blue, amino acid residues whose mutation disrupts DARC binding in red, basic

residues in blue and clusters of polymorphic residues in grey. c, GRASP representation

of Pka-DBL electropotential surface with positive (blue) and negative (red) patches.

Residues proximal to the DARC-binding site (Lys 96, Lys 100, Arg 103 and Lys 177)

form a continuous patch of electropositive density (blue).

42

0.7

0.6

e o.s

c: N 0.4

! • 0.3

0 0 0.2

0.1

0


Pv-OBL Pka-DBL Pka-DBL EBA175 AMA-1

Figure 2. 7: Binding specificity of recombinant Pkcx-DBL to humanDARC

2.3 Discussion:

Here, we have solved the structure of Pka-DBL from the duffy-binding protein a

of P. knowlesi. The overall structures of the Pka-DBL domain and the previously solved

EBA-175 region II DBL domains (F1 and F2) are very similar. The disulfide linkages in

each of these DBL domains are also identical except that F2 of EBA-175 has an

additional pair of cysteines that form a unique disulfide linkage in subdomain 3. Despite

having similar overall structures, the oligomeric states and binding sites of EBA-175

region II and Pka-DBL domain are strikingly different. EBA-175 region II forms

dimers; the Pka-DBL domain is monomeric. The amino acid stretches in F1 and F2 DBL

domains that serve as dimerization elements are missing in Pka-DBL and PvRII

domains (Figure 2.4 c). The Pka-DBL domain and PvRII have a single binding site for

DARC that is exposed fully. In contrast, the EBA-175 region II dimer contains six sialic

acid-binding sites, of which four reside in channels and two are surface exposed. Unlike

the interactions between PvRII and Pka-DBL with DARC, engagement of sialic acids on

glycophorin A by EBA-175 could require significant conformational rearrangements in

43


the E:SA-175 region II dimer. This comparative analysis highlights the diverse ways in

which conserved DBL domains can interact with their respective receptors.

Although extensive sequence polymorphisms in PvRII have been found in P.

vivax field isolates (Tsuboi et al. 1994; Ampudia et al. 1996; Xainli et al. 2000), the

contact residues that form the DARC-recognition site within PvRII appear to be invariant

(VanBuskirk et al. 2004; Hans et al. 2005). Analysis of sequence-polymorphism data

from diverse geographical locations (Papua New Guinea and Colombia) suggests that

there are two contiguous .. stretches of amino acids within PvRII (from 123 to 149 and 247

to 269) enriched in polymorphic residues (>40% ). These polymorphic clusters lie on the

surface opposite to the DARC-recognition site (Figure 2.8), suggesting that naturally

acquired antibody responses are directed predominantly against regions distal to the

proposed recognition site. In addition to the polymorphic clusters, isolated amino acid

polymorphisms are also found in PvRII. The majority of such point polymorphisms also

lie distal to the DARC recognition site. Only four of the reported polymorphic residues in

PvRII lie in the vicinity of the DARC-recognition site. Of these, Leu87 and Cys99 are

buried and are therefore unlikely to have arisen in response to immune pressure. D97V

has only been observed once in the field. The fourth polymorphic residue, Asn171, is

replaced frequently with Lys, which is also the case in Pka-DBL domain. It is not clear

whether this polymorphism can be attributed to immune pressure.

While several studies have mapped the DARC-recognition site on PvDBL or

Pka-DBL domains to ~ubdomain 2, the functional role of subdomain 3 has remained

unclear. The a7 and a9 helices that constitute the helical towers of subdomain 3 display

structural similarity to the fusion-active state of the 41 kDa ectodomain of HIV surface

glycoprotein (HIV gp41), as determined by DALI (Holm and Sander 1996). Whether the

a7 and a9 helices of PvDBL domain have a functional role in invasion similar to HIV

gp41 helices remains to be determined. Subdomain 3 loop adjoins the three highly

conserved disulfide bonds, where the disulfide pairs between Cys7-Cys9 and Cys8-Cys12

fix it on top of a7 and a9, such that binding of DARC with the binding site in subdomain

2 will juxtapose this segment proximal to the host membrane (Figure 2.9). Given the

invariant nature of disulfide bonds at the top of subdomain 3, fixed orientation of this

loop and conservation of Lys217, subdomain 3 might be involved in post receptor

44


recognition events that lead to junction formation. The function of subdomain 3 in

invasion remains to be explored experimentally.

Figure 2 .8 : Molecular-surface representation of PkaDBL

domain. (a),(b) Molecular-su1jace representation of Pka- DBL domain (grey) is

shown using the CHIMERA program. The figure highlights DARC recognition residues

(orange), polymorphic clusters (pink) and isolated polym01phic residues (green). (b) is

derived by 18rf rotation of (a) in the horizontal plane as indicated by the arrow.

The 'just-in-time' secretion of Pv-EBP from the nucronemes ts probably

immediately followed by DARC engagement, thus burying the binding site residues such

that they remain protected from any immune response against them. In this model (Figure

2.9) residues on the surface opposite to DARC recognition site would be exposed to

immune surveillance which is where polymorphism is predominantly present, possibly in

order to escape immune clearance. This parasite strategy to escape detection is essentially

45


different from that used by HIV and influenza. HIV obscures receptor binding residues

on gp120 by glycosylation/conformational camouflaging, (Wyatt eta!. 1998; Kwong et

a!. 2002) while in influenza virus, sequence polymorphisms in hemagglutinin are

concentrated around the active site pocket (Wilson and Cox 1990). The distal placement

of naturally occurring sequence polymorphisms from DARC binding site in Pv-DBL

qualifies this protein for its use as a vaccine against P. vivax malaria, since antibody

response against recombinant Pv-DBL is unlikely to be affected by field polymorphisms.

The structure of Pka-DBL may therefore provide a platform for the development of

inhibition strategies against P. vivax invasion of human erythrocytes.

Figure 2.9: Model of Pv-DBL-erythrocyte interaction.

Molecular surface showing PvDBL clusters of polymorphic residues (grey) and the

DARC recognition site (orange). Subdomains 1, 2 (blue) and 3 (green) are shown. Note

the position of the subdomain 3 loop (yellow). Docking of subdomain 2 onto DARC

would bring the subdomain 3 loop close to the host erythrocyte membrane (red

rectangle).

Owing to redundant P. falciparum erythrocyte invasion pathways, the interaction

between EBA-175 and glycophorin A is dispensable (Reed et a!. 2000). Therefore, in

46


order to block multiple P. falciparum invasion routes, EBA-175 and its homologues must

be targeted simultaneously. In contrast, Pv-DBL: DARC interaction is essential for P.

vivax invasion of human erythrocytes, making it an attractive target for therapeutic

strategies. Trw crtpu:xtupaA. avaA.'Jfmcr o<j> nm-~BNTIKa-DBL provides a platform

for development of inhibitory agents.

47

Documents

Structure q[Pka-DBL - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/16834/9/09... · 2018-07-09 · Structure of Pka-DBL 2.0 Introduction Malaria pathogenesis is mediated by