An imala ial Ac i i y o Cup edoxins
THE INTERACTION OF PLASMODIUM MEROZOITE SURFACE PROTEIN 1
19
(MSP1
19
)AND
RUSTICYANIN
*
Recei ed o publica ion, Feb ua y 14, 2013, and in e ised o m, June 7, 2013 Published, JBC Pape s in P ess, June 7, 2013, DOI 10.1074/jbc.M113.460162
Isabel C uz-Galla do
‡1
, I ene Díaz-Mo eno
‡
, An onio Díaz-Quin ana
‡
, An onio Donai e
§2
,
Ad ián Velázquez-Campoy
¶3
, Rachel D. Cu d
储4
, Ka e i Rangacha i
储
, Be y Bi dsall**, And es Ramos**
5
,
An hony A. Holde
储6
, and Miguel A. De la Rosa
‡7
F om he
‡
Ins i u o de Bioquímica Vege al y Fo osín esis (IBVF), cicCa uja, Uni e sidad de Se illa-CSIC, A enida Amé ico Vespucio
49, Se illa 41092, Spain, he
§
Depa amen o de Química Ino gánica, Facul ad de Química, Uni e sidad de Mu cia, Campus
Uni e si a io de Espina do, Mu cia 30100, Spain, he
¶
Ins i u o de Biocompu ación y Física de Sis emas complejos (BIFI),
Uni e sidad de Za agoza, c/Ma iano Esquillo , Za agoza 50018, Spain, he
储
Pa asi ology Di ision and **Molecula S uc u e
Di ision, Medical Resea ch Council (MRC) Na ional Ins i u e o Medical Resea ch, The Ridgeway, Mill Hill,
London W7 1AA, Uni ed Kingdom
Backg ound: The in e ac ion o MSP1
19
wi h he cup edoxin azu in inhibi s he g ow h o Plasmodium alcipa um in ed
blood cells.
Resul s: Rus icyanin o ms a well de ined complex wi h MSP1
19
upon binding a he same su ace a ea han inhibi o y
an ibodies.
Conclusion: Rus icyanin becomes an excellen he apeu ic agen o mala ia.
Signi icance: Knowing he us icyanin-MSP1
19
in e ace will allow he design o no el an imala ial d ugs.
The disco e y o e ec i e new an imala ial agen s is u gen ly
needed. One o he mos equen ly s udied molecules ancho ed
o he pa asi e su ace is he me ozoi e su ace p o ein-1
(MSP1). A ed blood cell in asion MSP1 is p o eoly ically p o-
cessed, and he 19-kDa C- e minal agmen (MSP1
19
) emains
on he su ace and is aken in o he ed blood cell, whe e i is
ans e ed o he ood acuole and pe sis s un il he end o he
in acellula cycle. Because a numbe o speci ic an ibodies
inhibi e y h ocy e in asion and pa asi e g ow h, MSP1
19
is
he e o e a p omising a ge agains mala ia. Gi en he s uc-
u al homology o cup edoxins wi h he Fab domain o mono-
clonal an ibodies, an app oach combining NMR and iso he mal
i a ion calo ime y (ITC) measu emen s wi h docking calcula-
ions based on BiGGER is employed on MSP1
19
-cup edoxin
complexes. Among he cup edoxins es ed, us icyanin o ms a
well de ined complex wi h MSP1
19
a a si e ha o e laps wi h
he su ace ecognized by he inhibi o y an ibodies. The addi-
ion o holo- us icyanin o in ec ed cells esul s in pa asi emia
inhibi ion, bu negligible e ec s on pa asi e g ow h can be
obse ed o apo- us icyanin and o he p o eins o he cup e-
doxin amily. These indings poin o us icyanin as an excellen
he apeu ic ool o mala ia ea men and p o ide aluable
in o ma ion o d ug design.
Mala ia is a widely sp ead disease causing mo bidi y and
mo ali y h oughou a la ge pa o he wo ld. The inc easing
esis ance o Plasmodium alcipa um, he causa i e agen o
he mos deadly o m o he disease, o cu en d ugs has only
inc eased he u gency o inding new an imala ial agen s (1, 2),
including an e ec i e accine and new d ug he apies (3, 4).
The e a e i e Plasmodium species ha in ec humans, whe eas
o he s in ec o he p ima es o oden s. Among his la e
g oup, oden pa asi es such as Plasmodium yoelii p o ide use-
ul labo a o y models o he s udy o mala ia. The disease is
caused by he eplica ion and mul iplica ion o he asexual
blood s ages in ed blood cells. The me ozoi e o m o he pa -
asi e in ades he hos cell, whe e i de elops and eplica es o
o m se e al new me ozoi es ha hen bu s ou o he cell o
con inue he cycle o in asion and mul iplica ion. The in asion
o ed blood cells equi es an ini ial ecogni ion and binding
media ed by pa asi e su ace ligands, ollowed by eo ien a ion
and he o ma ion o a mo ing junc ion be ween he e y h o-
cy e and me ozoi e su aces as he pa asi e en e s he cell.
Me ozoi e su ace p o ein-1 (MSP1)
8
has been implica ed in
his ini ial binding be ween pa asi e and hos cell.
Loca ed on he su ace o he asexual blood-s age schizon
and me ozoi e, MSP1 is one o he mos equen ly s udied
molecules o he pa asi e (5). I is syn hesized as a ⬃200-kDa
*This wo k was suppo ed in pa by Jun a de Andalucía G an s P08-CVI-3876
and BIO198 (Spain) and Eu opean Social Fund-ERDF 2007–2013.
1
Suppo ed by Jun a de Andalucía Ph.D. G an P08-CVI-3876 and GERMN
bu sa ies o sho e m s ays (Spain).
2
Suppo ed by Minis e io de Economía y Compe i i idad G an SAF2011-
26611 and Fundación Séneca de la Región de Mu cia G an 15354/PI/10
(Spain).
3
Suppo ed by Minis e io de Ciencia e Inno ación G an BFU2010-19451
(Spain).
4
Recipien o a Medical Resea ch Council s uden ship.
5
Suppo ed by Medical Resea ch Council G an U117574558.
6
Labo a o y wo k suppo ed by Medical Resea ch Council G an U117532067
and he Eu opean Union FP7 Ne wo k o Excellence E iMala .
7
To whom co espondence should be add essed. Tel.: 34-954489506; Fax:
34-954460065; E-mail: [email p o ec ed].
8
The abb e ia ions used a e: MSP1, me ozoi e su ace p o ein-1; Az, azu in;
BiGGER, bimolecula complex gene a ion wi h global e alua ion and ank-
ing; HSQC, he e onuclea single-quan um cohe ence; ITC, iso he mal i a-
ion calo ime y; mAb, monoclonal an ibody; MSP1
19
, MSP1 19-kDa C- e -
minal agmen ; Pc, plas ocyanin; PDB, P o ein Da a Bank; Rc, us icyanin.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 29, pp. 20896–20907, July 19, 2013
© 2013 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. Published in he U.S.A.
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p ecu so a ached o he su ace o he pa asi e ia a glycosyl-
phospha idylinosi ol ancho , which unde goes a wo-s ep p o-
eoly ic p ocess: i s , a me ozoi e elease and hen a e y h o-
cy e in asion (6). As a esul o his p ocessing, he MSP1 is
clea ed in o se e al polypep ides ha a e shed om he su ace
in he inal p ocessing s ep, sa e a 19-kDa C- e minal agmen
(MSP1
19
). MSP1
19
is e ained on he pa asi e su ace by he
glycosylphospha idylinosi ol ancho and aken in o he ed
blood cell a in asion (7–9). The ole o MSP1
19
in he subse-
quen in acellula de elopmen o he pa asi e is poo ly unde -
s ood, al hough i is ans e ed o he de eloping ood ac-
uole, whe e i emains un il he end o he in acellula cycle
and is disca ded in he esidual body oge he wi h p oduc s
o hemoglobin diges ion such as hemozoin (10). MSP1
19
is
conside ed a p omising mala ia accine candida e due o he
abundan e idence o speci ic an ibodies inhibi ing e y h o-
cy e in asion and pa asi e g ow h, o ins ance, ia he dis-
up ion o MSP1 p o eoly ic p ocessing and in acellula
pa asi e de elopmen (11).
A he s uc u al and unc ional le els, MSP1
19
is pa icula ly
well conse ed among Plasmodium species (Fig. 1) (12–17),
and i s h ee-dimensional s uc u e has been shown o consis
o wo epide mal g ow h ac o (EGF)-like domains in close
con ac . A cha ac e is ic disul ide-b idge pa e n (Fig. 1) makes
MSP1
19
highly esis an o p o eases (19) and may explain why
MSP1
19
emains in ac in he diges i e ood acuole up o he
end o he in acellula cycle (10).
MSP1-speci ic immunoglobulins eac wi h con o ma ional
epi opes o MSP1
19
. Some o hese an ibodies inhibi pa asi e
in asion o e y h ocy es, whe eas o he s do no . Fine s uc u e
epi ope mapping o di e en monoclonal an ibodies (mAbs)
and he use o NMR me hods indica es he binding o wo
inhibi o y an ibodies o epi opes on one side o he molecule
nea he in e ace be ween he wo EGF domains, including
esidues om bo h domains (20, 21). By con as , non-inhibi-
o y neu al mAbs bind elsewhe e on he molecule (15, 20).
He e, we ha e used MSP1
19
om P. yoelii, a oden mala ia
pa asi e used as a labo a o y model o accine s udies (22)
and o which bo h inhibi o y and neu al an ibodies ha e
been pa ially mapped on he s uc u e (23, 24).
9
Independ-
en ly o he immunoglobulin class, complexes in ol ing
MSP1
19
a e kine ically a he s able wi h dissocia ion con-
s an s in he mic omola o subnanomola ange (15, 25).
10
Because he binding a ini y is simila in all cases, i has been
assumed ha he inhibi o y e ec depends mainly on s e ic
ac o s, namely, epi ope loca ion, a he han he na u e o
he an ibody.
The p esen s udy has been based on he s uc u al homology
o cup edoxins wi h he Fab agmen o an an ibody, as well as
on epo s o a p o ein om his amily in e ac ing di ec ly wi h
MSP1
19
and blocking he inc ease o pa asi emia in human ed
blood cells in ec ed by P. alcipa um, sugges ing a p omising
ea men (26, 27). We i s used he DaliLi e pai wise compa -
ison p og am o iden i y s uc u al simila i ies be ween cup e-
doxins and he Fab agmen (28). A sc eening was hen pe -
o med combining NMR and ITC measu emen s wi h docking
calcula ions using BiGGER which indica ed ha , among he
cup edoxins es ed, us icyanin (Rc) p o ided he mos e ec-
i e binding o MSP1
19
. The wo p o eins o m a well de ined
complex whe e Rc in e ac s a he in e ace be ween he wo
MSP1
19
subdomains, a a si e ha o e laps wi h he su ace
ecognized by he inhibi o y an ibodies. Fu he , P. alcipa um
g ow h is inhibi ed by he p esence o Rc in ed blood cell cul-
9
R. D. Cu d and A. A. Holde , unpublished da a.
10
M. J. Lock and A. A. Holde , unpublished da a.
FIGURE 1. MSP1
19
p o ein. A, mul iple sequence alignmen o MSP1
19
wi h Clus alW2 (18) om a numbe o Plasmodium species. Sequences a e colo ed by
pe cen iden i y: black (100%), da k g ay (80%), ligh g ay (60%), and whi e (⬍50%). Seconda y s uc u e elemen s o P. yoelii MSP1
19
a e shown. O ange boxes
highligh cys eine esidues ha a e conse ed ac oss di e en Plasmodium species, whe eas ed boxes highligh hose p esen only in P. alcipa um.Gold a ows
s and o disul ide bonds o med by cys eine esidues. The main di e ence be ween P. alcipa um MSP1
19
and homologs om o he Plasmodium species
cha ac e ized o da e lies in he disul ide bond pa e n o he p o ein om he o he species: one o he cys eine pai s in he i s EGF domain is subs i u ed by
a yp ophan and a nonpola o alipha ic esidue. B, ibbon ep esen a ion o he lowes ene gy NMR s uc u e o P. yoelii MSP1
19
(BMRB accession numbe :
19233). Side chains o cys eine esidues o ming disul ide b idges a e ep esen ed in gold.
MSP1
19
-Rus icyanin Complex
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u es. In e es ingly, he coppe si e plays a key ole in complex
o ma ion, because apo-Rc is no only unable o in e ac wi h
MSP1
19
, bu also o inhibi pa asi e in asion and de elopmen
in in ec ed ed blood cells.
EXPERIMENTAL PROCEDURES
Exp ession and Pu i ica ion o P o eins—P. yoelii MSP1
19
(
15
N-labeled o unlabeled) was p oduced (essen ially as
desc ibed p e iously (29)) om a syn he ic gene op imized o
Pichia pas o is exp ession using as ni ogen sou ce ei he
15
NH
4
Cl o (NH
4
)
2
SO
4
o labeled and unlabeled p o ein,
espec i ely. The 99-amino acid sequence co esponds o esi-
dues 1656–1754 o he UniP o KB en y P13828 con aining he
N- e minal ag HHHHHHIEGR ha has li le e ec on he
NMR spec um (20). Sec e ed His
6
- agged MSP1
19
was pu i ied
om he cul u e medium by nickel a ini y ch oma og aphy
(Ni-Sepha ose 6 Fas Flow; GE Heal hca e) and acco ding o a
p e iously elabo a ed p o ocol (12).
All ecombinan me allop o eins we e exp esed in Esche-
ichia coli cul u es in LB medium and pu i ied acco ding o
p e iously elabo a ed p ocedu es, namely Acidi hiobacillus e -
ooxidans Rc (30, 31), Nos oc sp. PCC 7119, Pho midium lami-
nosum and popla plas ocyanins (Pc) (32–34) and Pseudomo-
nas ae uginosa azu in (Az) (35).
NMR Spec oscopy—All p o ein samples we e concen a ed
in 10 mMpo assium phospha e (pH 6.5) using Millipo e 3000
NMWL cen icons and mic ocons. MSP1
19
samples anged in
concen a ion om 0.5 o 2 mM, whe eas cup edoxins we e
used in he ange o 2–5 mM. All NMR samples con ained 10%
D
2
O o adjus he lock signal. Reduc ion o he me al cen e in
samples o coppe (I) cup edoxins was achie ed by adding
sodium asco ba e, whe eas oxida ion o he me al cen e in
samples o coppe (II) p o eins was achie ed using sodium e -
icyanide o Pc and Az, and sodium hexachlo oi ida e(IV) o
Rc. In all cases, he p o eins we e washed ex ensi ely o emo e
he excess o he educing/oxidizing agen .
NMR expe imen s we e pe o med in a B uke A ance 600
MHz spec ome e a 25 °C. The sequence-speci ic assignmen
o he backbone amide g oups o
15
N MSP1
19
(BMRB accession
numbe : 19233) was achie ed using s anda d backbone expe -
imen s (HNCACB, HNCA, e c.) and was con i med using
h ee-dimensional
1
H-
15
N NOESY-HSQC and h ee-dimen-
sional
1
H-
15
N o al co ela ion spec oscopy-HSQC spec a.
The in e ac ion o MSP1
19
wi h cup edoxins was ollowed by
acqui ing wo-dimensional
1
H-
15
N HSQC spec a du ing he
i a ion o 0.5 mM
15
N-MSP1
19
solu ions wi h an inc easing
amoun o oxidized o educed cup edoxins up o a inal cup e-
doxin:MSP1
19
mola a io o 4:1. The pH alue o he sample
was e i ied a e each i a ion s ep. P olines, which a e in is-
ible esonances in
15
N HSQC spec a, a e loca ed a he posi-
ions 4, 15, 50, 61, 84, and 86, whe eas Gly-1, Val-2, Glu-69, and
Asn-73 a e unassigned esidues. All da a p ocessing was pe -
o med wi h B uke TopSpin 2.0, and NMR analysis o line
b oadening pe u ba ions o he cup edoxin-bound MSP1
19
wi h espec o ee mala ial p o ein was pe o med in he
SPARKY p og am (36).
NMR Line Wid h Analysis—To es ima e line wid hs, he
peaks we e i ed o a Gaussian unc ion o he
15
N and
1
H
dimensions using he p og am SPARKY wi h a 10,000 s eps
minimiza ion and a 0.05% ole ance. In he analysis o he line
wid hs (⌬
␯
1/2
), he o e all b oadening (⌬⌬
␯
1/2
) ob ained om
signals displaying only mino line b oadening was i s sub-
ac ed om he line wid h o he co esponding signal. Then,
o each esidue, he di e ences o line wid hs be ween ee
and in e ac ing MSP1
19
we e calcula ed in e e y i a ion
se ies (⌬⌬
␯
1/2 Binding
). The h eshold alue, used o iden i y a
speci ically b oadened esidue when da a om he i a ion se ies
we e analyzed oge he , was de ined as he a e age ⌬⌬
␯
1/2 Binding
o he sys em plus 2 s anda d de ia ions (2S
n⫺1
). The a e -
age ⌬⌬
␯
1/2 Binding
and s anda d de ia ion we e calcula ed o
all amides wi h alues ⱕ10 Hz on he basis ha da a ⬎10 Hz
clea ly indica ed a speci ically b oadened esidue, and hei
inclusion would bias he a e age o a highe alue. Some
assigned signals o he ee MSP1
19
HSQC spec um o e lap
(Val-9, Asn-87, and Cys-95) o exhibi e y low in ensi y
(Gly-41 and Asn-42), so hey could no be p ope ly in eg a ed
o include hem in he line wid h analysis.
ITC—All ITC expe imen s we e pe o med using VP-ITC
and Au o-ITC200 ins umen s (Mic ocal; GE Heal hca e) a
25 °C i a ing Rc wi h MSP1
19
. The e e ence cell was illed
wi h dis illed wa e . The expe imen s consis ed o 10-
␮
lo 2-
␮
l
injec ions o 0.3 mMMSP1
19
solu ion in 10 mMpo assium phos-
pha e bu e (pH 6.5) in o he sample cell, ini ially con aining
6.67
␮
MRc solu ion ( educed, oxidized, and apo o ms) in he
same bu e . All o he solu ions we e degassed be o e he i a-
ions we e pe o med. Ti an was injec ed a app op ia e ime
in e als o ensu e he he mal powe signal e u ned o he
base line p io o he nex injec ion. To achie e homogeneous
mixing in he cell, he s i ing speed was kep cons an a 1000
pm in he Au o-ITC200 and a 450 pm in he VP-ITC. The
da a, speci ically he hea pe injec ion no malized pe mol o
injec an e sus mola a io, we e analyzed wi h O igin 7
(Mic ocal) using a single-si e binding model. Calib a ion and
pe o mance es s o he calo ime e we e ca ied ou conduc -
ing CaCl
2
-EDTA i a ions wi h solu ions p o ided by he
manu ac u e .
Molecula Docking Simula ions—A so docking algo i hm
implemen ed in he BiGGER so wa e package (37) was used o
de e mine in silico a model o he complexes MSP1
19
-Az,
MSP1
19
-Pc, and MSP1
19
-Rc. The PDB coo dina es iles o
cup edoxins we e 1JZG o Az (38), 1NIN o Pc (39), and 1A3Z
o Rc (40). Fo each un, 5000 docking geome ic solu ions
we e gene a ed based on he complemen a i y o he p o ein
su aces. These solu ions we e e alua ed and anked acco ding
o hei “global sco e” and di e en in e ac ion c i e ia includ-
ing elec os a ic ene gy o in e ac ion, ela i e sol a ion ene gy,
and he ela i e p opensi y o side chains o in e ac . Fo he
MSP1
19
-Rc adduc , NMR es ain s we e in oduced in he
docking calcula ions. All complexes g aphic images we e gen-
e a ed using he UCSF Chime a package (41).
P. alcipa um Cul u es—Synch onized P. alcipa um 3D7
la e s age ophozoi es a 33–36 h we e used. The inal pa asi -
emia and hema oc i we e be ween 0.1–0.2 and 2%, espec-
i ely. Red blood cells used o he assay we e cen i uged o
emo e he bu y coa and washed wice in RPMI 1640
medium so ha no whi e blood cells we e p esen . The cul-
MSP1
19
-Rus icyanin Complex
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u e medium con ained RPMI 1640 medium supplemen ed
wi h 5 g/li e albumax, 0.025 g/li e gen amycin, and 0.292
g/li e L-glu amine.
P. alcipa um G ow h Inhibi ion Assay—S e ile 96-well black
issue cul u e pla es (Cos a ) we e used ou inely o e e y
assay. The holo- o apo-species o Rc and Nos oc Pc we e
dilu ed in cul u e medium and used in duplica e wells o each
dilu ion (200, 100, 50, and 25
␮
M, espec i ely) in a inal olume
o 100
␮
l/well. Two con ol se s we e used in duplica e wells,
one se wi h no added cup edoxin (posi i e con ol) and one
wi h unin ec ed ed blood cells (nega i e con ol). The pla es
we e incuba ed a 37 °C o 48 h in a gas chambe lushed wi h
5% CO
2
,5%O
2
, and 90% N
2.
A e 48 h, supe na an s we e
emo ed om each well, eplaced wi h esh medium con ain-
ing p o ein, and incuba ed o a u he 48 h in he same man-
ne . A he end o he 96-h incuba ion, 25
␮
l o SYBR G een I
dye (SYBR G een I nucleic acid gel s ain 10,000⫻, in dime hyl
sul oxide om In i ogen) in lysis bu e (1
␮
l dye o 1 ml lysis
bu e ), was added o each well and s o ed o e nigh a ⫺20 °C.
The lysis bu e con ained T is-HCl (20 mM, pH 8.0), EDTA (2
mM), Saponin (0.16% w/ ) and T i on X-100 (1.6% / ). Pla es
we e hawed a oom empe a u e, and luo escence in ensi y
was measu ed wi h a FLUO S a Omega mic opla e luo es-
cence eade (BMG Lab ech). Values we e exp essed in ela i e
luo escence uni s. Binding o SYBR G een is speci ic o pa a-
si e DNA as ma u e e y h ocy es lack DNA and RNA. Fluo es-
cence in ensi y uni was con e ed o pe cen age (%) o g ow h
as ollows: % g ow h ⫽(cul u e unde Rc o Pc) ⫺(unin ec ed
RBC)/(cul u e wi h no Rc o Pc) ⫺(unin ec ed RBC) ⫻100,
whe e RBC a e ed blood cells.
FIGURE 2. Conse ed s uc u al mo i s. A, ibbon ep esen a ion o he Fab agmen o mAb G17.12 (PDB ID code 1OB1). Domain A is depic ed in ligh g ay
and domain B in gold.B, s uc u al alignmen o Az (uppe , PDB ID code 1JZG) and A1 agmen o Fab (lowe ) buil by DaliLi e pai wise compa ison se e .
Ma ching egions, wi h a Dali Z-sco e o 2.9, appea in o ange.C, ibbon ep esen a ion o Rc (uppe , PDB ID code 1A3Z) and Pc (lowe , PDB ID code 1NIN). Dali
Z-sco es o Rc and Pc a e 2.6 and 3.1, espec i ely. Coppe a oms a e colo ed in blue.
FIGURE 3. Ab ini io docking o cup edoxins wi h MSP1
19
pe o med by BiGGER. Le , Az. Cen e , Pc. Righ , Rc. Ligh yellow sphe es ep esen he geome y
cen e s o MSP1
19
in he 100 bes solu ions gene a ed o each complex. All cup edoxins a e o ien ed wi h espec o hei coppe cen e . Robe son diag ams
o he cup edoxins a e colo ed acco ding o seconda y s uc u es:
␣
-helices in ed and
␤
-s ands in blue. Coppe a oms a e ep esen ed as blue sphe es and he
esidues bonding o hem a e in g ay s icks.
MSP1
19
-Rus icyanin Complex
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RESULTS
S uc u al Simila i ies be ween Cup edoxins and he Fab
F agmen o a mAb—The Fab agmen c ys al s uc u e o
mAb G17.12 (15), shown o bind MSP1
19
, is composed o wo
domains, A and B, con aining wo egions, a iable (symbolized
by 1) and cons an (symbolized by 2), wi h he canonical
␤
-sandwich old o he immunoglobulin supe amily (Fig. 2A).
Consis en wi h p e ious epo s, he DaliLi e pai wise com-
pa ison p og am (28) iden i ied signi ican s uc u al simila i-
ies be ween he A1 agmen o he mAb and coppe -con ain-
ing edox p o eins wi h immunoglobulin old, such as Az, Pc,
and Rc. In pa icula , he s uc u al alignmen be ween he A1
agmen and Az e eals a ma ching egion, which is localized
mainly a he wo an ipa allel
␤
-shee s, as desc ibed p e iously
(26; Fig. 2B), and wi h a Dali Z-sco e o 2.9. Fo he wo o he
cup edoxins, Pc and Rc, his s uc u al ma ch is ex ended o he
loops connec ing
␤
-s ands yielding Z-sco es o 3.1 and 2.6,
espec i ely (Fig. 2C).
Gi en he s uc u al simila i y be ween a ious cup edoxins
and he Fab agmen , ab ini io docking app oaches we e pe -
o med wi h no expe imen al es ain s (Fig. 3) o explo e he
capabili y o he me allop o eins o in e ac wi h MSP1
19
om
P. yoelii. MSP1
19
is well conse ed among he species wi h
⬎50% sequence iden i y (Fig. 1A), wi h conse ed h ee-di-
mensional s uc u e and common unc ional ea u es (17). We
used he BiGGER igid docking algo i hm o gene a e se s o
possible o ien a ions o he di e en cup edoxin p obes
a ound MSP1
19
( he a ge ). Fig. 3 shows he dis ibu ion o
MSP1
19
mass cen e s esul ing om he 100 bes solu ions
om each compu a ion a ound he co esponding coppe p o-
ein. Fo Az and Pc, we obse e a ema kably b oad dispe sion
o he MSP1
19
geome y cen e s, sugges ing he lack o speci ic
su ace complemen a i y. By con as , molecula docking o
he Rc-MSP1
19
in e ac ion indica es ha MSP1
19
explo es a
well de ined a ea o Rc su ounding i s coppe cen e .
MSP1
19
In e ac ions wi h Cup edoxins by NMR—The
MSP1
19
-cup edoxin in e ac ion was moni o ed by eco ding
wo-dimensional
15
N HSQC NMR spec a on
15
N-MSP1
19
,
bo h ee and ollowing he addi ion o Az, Pc, o Rc. The
absence o changes in MSP1
19
esonances, ei he chemical shi
pe u ba ions o line b oadening, indica es no de ec able bind-
ing o Az and Pc in any o hei oxida ion s a es (Fig. 4, op and
middle panels). By con as , line wid h changes o ce ain
MSP1
19
amide signals upon addi ion o Rc(Cu
⫹
) sugges a spe-
ci ic MSP1
19
-Rc in e ac ion (Fig. 4, bo om panel). Such obse -
a ions coincide wi h ab ini io docking simula ions pe o med
(Fig. 3) which sugges ed a well de ined complex only be ween
Rc and MSP1
19
.
To p obe in g ea e de ail he in e ac ion o MSP1
19
wi h
ei he oxidized o educed Rc, we analyzed he line wid hs o
MSP1
19
esonances om
15
N HSQC spec a ac oss se e al
i a ions. Binding o Rc esul s in gene al signal b oadening
due o he inc ease in he o a ional co ela ion ime o MSP1
19
when in e ac ing wi h Rc. In addi ion, se e al MSP1
19
back-
bone amides clus e ed in one a ea o he s uc u e unde go
la ge changes in line wid hs (⌬⌬
␯
1/2 Binding
) upon Rc binding
(Figs. 5 and 6). These esonances a e expec ed o be a o in he
p oximi y o he a ea o MSP1
19
in e ac ing wi h Rc. To selec-
i ely de ine esonances mos likely o be pa o he in e ace,
h eshold alues (speci ically, ⌬⌬
␯
1/2 Binding
ⱖ5Hz o
15
N
and ⱖ11Hz o
1
H dimension) we e se (see “Expe imen al
P ocedu es”). As expec ed, he dis ibu ion o line wid h
changes (⌬
␯
1/2
) becomes b oade as he Rc:MSP1
19
a io
inc eases (da a no shown).
FIGURE4. NMR i a ionso
15
NMSP1
19
wi h educed cup edoxins.Top,Az
om P. ae uginosa.Middle, Pc om Nos oc sp. PCC 7119. Bo om, Rc om A.
e ooxidans. Supe imposi ion o
15
N HSQC spec a o ee MSP1
19
(magen a)
and a e adding one o h ee cup edoxins (blue) a a cup edoxin:MSP1
19
a io
o 4:1 is shown. A ows poin ou hose esidues ha expe ience subs an ial
b oadening. Negligible binding o MSP1
19
was obse ed upon adding ali-
quo s o Pc and Az.
MSP1
19
-Rus icyanin Complex
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A he Rc(Cu
1⫹
):MSP1
19
a io o 1.5:1, 14 amino acids show
conside able
15
N line wid h changes in MSP1
19
HSQC spec a
(Fig. 5, Aand B). These esidues a e dis ibu ed mainly along
wo egions o MSP1
19
, namely, a he beginning o N- e minal
EGF domain in ol ing Asp-3, Lys-5, His-6, Val-7, Asp-10, and
a he end o C- e minal domain comp ising Th -85, Ala-88,
Ty -89, Phe-94, and Se -97. In addi ion, Asp-24, Asp-25, and
A g-31 a he wo i s an ipa allel
␤
-s ands, along wi h Cys-
59, a e also al e ed. Fig. 5Cshows he map o MSP1
19
esidues
a ec ed by Rc addi ion, wi h colo s co esponding o line
b oadening in he
15
N dimension. The sequen ial s e ches o
esidues de ailed abo e o m he main clus e on he MSP1
19
su ace, which su ounds he wo EGF domain in e ace. In e -
es ingly, some o hese a ec ed esidues (Asp-24, Asp-25,
A g-31 and Cys-59) a e loca ed a he ea o he p o ein (Fig.
5C). Whe eas Asp-24 and Asp-25 a e close o amino acids a he
N e minus, A g-31 and Cys-59 lie nea Phe-94. Because hese
ou esidues a e adjacen o o he s placed a he EGF domain
in e ace and in ol ed in di ec con ac wi h Rc, hese line
wid h pe u ba ions a e p obably a seconda y e ec o binding.
Simila conclusions may be in e ed om
1
H line wid h analy-
sis, al hough p o ons a e mo e sensi i e o b oadening (da a no
shown).
NMR i a ion o oxidized Rc on MSP1
19
esul s in signi i-
can MSP1
19
line wid h pe u ba ions a a Rc(Cu
2⫹
):MSP1
19
a io o 4:1, a which some signals b oaden beyond he de ec-
ion limi (Fig. 6A). The MSP1
19
esonances a ec ed by
Rc(Cu
2⫹
) binding in ol e bo h N- e minal (Asp-3, Lys-5, Asp-
10, and Asp-13) and C- e minal (Cys-79, Th -85, Ty -89, Gly-
92, Phe-94, Se -97, and Se -98) egions, as desc ibed o he
educed sys em a a Rc(Cu
1⫹
):MSP1
19
a io o 1.5:1. An addi-
ional s e ch (Phe-21 o Asp-24, Gly-26, Glu-29, A g-31, and
Cys-59) is also pe u bed whe eas His-6, Val-7, Asp-25, and
Th -27 we e esidues o e he de ec ion limi (Fig. 6A). No a-
bly, he s eng h o he b oadening obse ed o a 4:1 Rc(Cu
2⫹
):
MSP1
19
a io is compa able wi h he one obse ed a a 1.5:1
Rc(Cu
1⫹
):MSP1
19
a io, indica ing ha MSP1
19
binds mo e
weakly o he oxidized Rc. Howe e , he MSP1
19
in e ac ing
su ace in ol ed in Rc ecogni ion a a Rc:MSP1
19
a io o 4:1 is
independen o he cup edoxin edox s a e (Fig. 6).
Binding o MSP1
19
o Holo-Rc by ITC—ITC measu emen s
e eal ha MSP1
19
binds o Rc ei he in i s educed o oxidized
s a e wi h a 1:1 s oichiome y a 25 °C (Fig. 7). No ably, he
in e ac ion o MSP1
19
wi h Rc(Cu
1⫹
) is exo he mic wi h a dis-
socia ion a ini y cons an (K
d
)o 2
␮
M, whe eas ha wi h
Rc(Cu
2⫹
) is an endo he mic p ocess wi h lowe binding a ini y
FIGURE 5. NMR i a ion o
15
N MSP1
19
wi h educed Rc. A,
15
N line wid h di e ences (
15
N⌬⌬
␯
1/2 Binding
) be ween ee and Rc-bound MSP1
19
. The Rc:MSP1
19
a ios a e 0.5:1 ( ed), 1:1 (g ay), and 1.5:1 (blue). B, supe posi ion o
15
N HSQC spec a o ee MSP1
19
(magen a) and bound o Rc (blue) in a Rc:MSP1
19
a io o
1.5:1. A subse o h ee ep esen a i e esonances is labeled in black.C, map o MSP1
19
in e ace upon binding o Rc. MSP1
19
su ace is o a ed 90° a ound he
e ical axes in each iew. Residues a e colo ed acco ding o hei
15
N⌬⌬
␯
1/2 Binding
(Hz): he esonances ha unde go he la ges b oadening (ⱖ5 Hz) a e
o ange, and he signals wi h a signi ican line wid h o e he de ec ion limi ⬍5Hza eyellow. The limi o 5 Hz co esponds o a h eshold alue ela i e o he
a e age plus 2- old he S.D. (⌬⌬
␯
1/2 Binding
ⱖ⬍⌬⌬
␯
1/2 Binding
⬎⫹2S
n⫺1
). Residues wi h no line wid h pe u ba ion a e ma ked in blue, whe eas p olines a e in
g ay.
MSP1
19
-Rus icyanin Complex
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(K
d
⫽25
␮
M) (Table 1). Such di e ences in K
d
alues a e in
ag eemen wi h NMR i a ions and sugges ha MSP1
19
binds
o educed Rc wi h a g ea e a ini y han o oxidized Rc.
Fu he expe imen s need o be pe o med o explain he
opposi e sign in he en halpy o bo h p ocesses as i could be
ela ed o (i) changes in he p o ona ion/dep o ona ion
equilib ium o ionizable g oups o he in e ac ing p o eins;
(ii) a ia ions in he hyd ogen-bonding ne wo ks; (iii) di e -
ences in wa e a angemen in he icini y o oxidized o
educed co ac o s; and (i ) sligh con o ma ional modi ica-
ions al e ing he numbe o sol en molecules excluded
om he p o ein in e ace.
FIGURE 6. Compa ison be ween NMR i a ions o
15
N MSP1
19
wi h ei he oxidized o educed Rc a Rc:MSP1
19
a io o 4:1. Mos o he MSP1
19
signals
in binding o Rc(Cu
1⫹
) a e b oadening o e he de ec ion limi a he Rc:MSP1
19
a io o 4:1, sugges ing ha MSP1
19
binds educed Rc wi h a highe a ini y. A,
uppe , o e lap be ween
15
N HSQC spec a o ee MSP1
19
(magen a) and oxidized Rc-bound MSP1
19
(blue). A,lowe , map o MSP1
19
in he p esence o oxidized
Rc. B, he same as Awi h educed Rc. The 90°- o a ed su ace ep esen a ions o MSP1
19
show esidues colo ed acco ding hei
15
N⌬⌬
␯
1/2 Binding
ollowing he
same colo code as in Fig. 5C. Those esidues b oadened beyond he de ec ion limi a e highligh ed in ed.
MSP1
19
-Rus icyanin Complex
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The Case o Apo-Rc—To de e mine he ole o he coppe
cen e , NMR and ITC i a ions we e ca ied ou using MSP1
19
and apo-Rc. Su p isingly, NMR i a ions showed no subs an-
ial line wid h changes e en a an apo-Rc:MSP1
19
a io o 4:1
(Fig. 8). The inding was u he co obo a ed by ITC measu e-
men s, as he weak calo ime ic p o ile sugges s a lack o in e -
ac ion (Fig. 8). Al oge he , hese da a indica e he ele ance o
he coppe cen e in he binding o MSP1
19
. Despi e he small
s uc u al di e ences in cup edoxins epo ed p e iously in
solid s a e, he e exis s a high deg ee o mobili y o he me al-
binding loops in solu ion in he apo o m, as ecen ly demon-
s a ed by NMR (42, 43). The di e en pa e n be ween apo-
and holo-Rc e sus MSP1
19
could be ela ed wi h he ound
di e ences in he dynamics o his si e in he wo o ms.
Docking Simula ions wi h BiGGER—Along wi h he ab ini io
docking calcula ions un on he MSP1
19
-Rc(Cu
⫹
) complex,
e ealing how he MSP1
19
mass cen e docks on he Rc me al
c e ice (Fig. 3), an NMR- es ained docking wi h BiGGER was
also pe o med. Line wid h da a o hose MSP1
19
esidues in
con ac wi h Rc a a Rc:MSP1
19
a io o 1.5:1 we e included in
he un. The ou pu is a se o docked solu ions ha can be
anked acco ding o he BiGGER global sco e o indi idual
sco es, such as hyd ophobic c i e ia, elec os a ics, and geo-
me ical pa ame e s. Fig. 9 shows he bes 100 solu ions, as
ep esen ed by Rc geome y cen e s, acco ding o he global
and hyd ophobic sco es om he p og am (Fig. 9A). The bes
sco ing models p edic ed by es ained docking e eal how
MSP1
19
leans i s EGF domain in e ace o app oach Rc (Fig.
9A). Indeed, he p oximi y o he su aces on bo h p o eins in
he complex is shown in he space- illing ep esen a ion (Fig.
9A). In addi ion o he coppe cen e , loops connec ing Rc
␤
-s ands a e in close con ac wi h MSP1
19
. A deep analysis o
he complex in e ace p edic ed by docking poin s o he N- e -
minal and C- e minal egions o he MSP1
19
as d i ing complex
o ma ion as some NMR es ain s a e sa is ied (Fig. 9B). In he
model epo ed, some elec os a ic in e ac ions occu (Fig. 9C)
because Rc esidues Lys-81 and Lys-116 a e close o Glu-91 and
Glu-83 in MSP1
19
. Fu he mo e, Lys-5 o MSP1
19
can in e ac
wi h Glu-9 o Rc, al hough he Glu is pa ially bu ied. The Rc
in e ac ion su ace is mainly hyd ophobic wi h he excep ion o
a single posi i e spo , Lys-81 and Lys-116, su ounded by Phe-
83, Gly-82, and T p-7 and he coppe cen e , which, in u n, is
enclosed by Me -99, Val-98, and P o-141. This in e ac ion
mode esembles hose o he coppe p o ein Pc and cy och ome
c
6
wi h hei pho osyn he ic pa ne s, cy och ome and pho o-
sys em I. In such complexes, Pc and cy och ome c
6
use he
hyd ophobic si e su ounding he coppe cen e and he heme
g oup (si e 1) as well as hei cha ged pa ch (si e 2; 44, 45).
Inhibi ion o P. alcipa um G ow h by Holo-Rc—To assess he
physiological ele ance o he MSP1
19
binding o Rc, he
g ow h o P. alcipa um wi hin ed blood cells was ollowed in
FIGURE 7. ITC i a ions o MSP1
19
wi h bo h edox s a es o Rc. Binding assays o he complexes be ween MSP1
19
and educed (le ) o oxidized Rc ( igh )
a e shown. The mog ams a e shown a he op and binding iso he ms a he bo om, along wi h he dissocia ion cons an (K
d
exp essed in
␮
M), en halpy (⌬H
in kcal/mol). The s oichiome y alue (n) was ixed o a oid degene acy in he nonlinea eg ession da a analysis. Typical ela i e e o s a e 20–25% o he
dissocia ion cons an and 5–10% o he s oichiome y.
TABLE 1
The modynamic alues in e ed om ITC expe imen s
The a ini y o a p o ein-p o ein in e ac ion is de ined by he Gibbs ene gy o he
binding: ⌬G⫽⫺RT ln K
a
.⌬Ghas wo di e en con ibu ions, ⌬Hand ⌬S, acco d-
ing o he equa ion: ⌬G⫽⌬H⫺T⌬S, hus se e al combina ions o hose alues
could yield simila binding a ini ies. The complex o ma ion is en opically d i en
in bo h cases, being he en halpic con ibu ion o he binding un a o able (15 kcal/
mol wi h oxidized Rc) o only sligh ly a o able (⫺1.7 kcal/mol wi h educed Rc).
MSP1
19
complex ⌬G⌬Hⴚ⌻⌬SK
d
kcal/mol kcal/mol kcal/mol
␮
M
Oxidized Rc ⫺6.3 15.0 ⫺21.3 25.0
Reduced Rc ⫺7.8 ⫺1.7 ⫺6.1 2.0
MSP1
19
-Rus icyanin Complex
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he p esence o ei he holo- o apo-Rc (see “Expe imen al P o-
cedu es”). The addi ion o holo-Rc o in ec ed cells esul ed in
pa asi emia inhibi ion, as cul u e g ow h dec eased signi i-
can ly (Fig. 10) a a concen a ion o 100
␮
Mun il he poin o
comple e inhibi ion a 200
␮
Mholo-Rc. In con as , he apo
o m e en a high concen a ions did no ha e a signi ican
e ec on P. alcipa um g ow h. These da a ully co obo a e
p e ious obse a ions using NMR o ITC as much as apo-Rc is
unable o bind o MSP1
19
.
To disca d he heo y ha such a pa asi emia inhibi ion
could be asc ibed o he well known apop o ic ole o se e al
cup edoxins (46, 47), con ol expe imen s we e un wi h he
holo- o apo- o m o Pc. Holo-Pc was chosen as i does no bind
o MSP1
19
, as in e ed om he he ein p esen ed NMR sc een-
ing (see abo e), bu i is s uc u ally e y simila o Rc: he wo
p o eins belong o he ype I blue coppe -p o ein amily, wi h
almos iden ical olding and e ahed al coppe cen e . Upon
addi ion o ei he holo- o apo-Pc (25
␮
M) unde he same cul-
u e condi ions, he Plasmodium g ow h i s sligh ly decays o
u he each a cons an alue un il he end o he expe imen .
So he Plasmodium g ow h does no depend on p o ein con-
cen a ion ( he pe cen age o g ow h is main ained a
app oxima ely 70% e en a 200
␮
MPc) and is p ac ically he
same wi h ei he holo- o apo-Pc. Al oge he hese esul s
indica e ha holo-Rc inhibi s pa asi emia upon speci ic
binding o MSP1
19
.
FIGURE 8. NMR and ITC i a ions o MSP1
19
wi h apo-Rc. A,
15
N line
wid h di e ences (
15
N⌬⌬
␯
1/2Binding
) be ween ee MSP1
19
and in he p es-
ence o apo-Rc. The apo-Rc:MSP1
19
a ios we e 1:1 ( ed), 1.5:1 (g ay), and
4:1 (blue). B, o e lap be ween
15
N HSQC spec a o ee MSP1
19
(magen a)
and in he p esence o apo-Rc (blue) a an apo-Rc:MSP1
19
a io o 4:1. C, ITC
he mog am ob ained om he apo-Rc-MSP1
19
i a ion, e ealing he
lack o binding be ween bo h p o eins because o he la calo ime ic
p o ile.
FIGURE 9. BIGGER molecula docking o he MSP1
19
-Rc complex. A,le ,
bes 100 models wi h he lowes ene gy alues a e alignmen o MSP1
19
molecules wi h Rc geome y cen e s ep esen ed by sphe es. Ribbon (cen e )
and space- illing ( igh ) ep esen a ions o he bes model a e shown in he
same o ien a ion as on he le . MSP1
19
is ep esen ed in da k g ay whe eas Rc
is in ligh blue.B, in e ace esidues o he MSP1
19
-Rc complex. MSP1
19
and Rc
a e independen ly o a ed 90° o he op and o he bo om, espec i ely,
wi h ega d o hei o ien a ion in A. Con ac ing esidues a e depic ed in ligh
yellow o he MSP1
19
and da k blue o Rc. C, elec os a ic po en ial su aces o
MSP1
19
(uppe ) and Rc (lowe ) wi h he same o ien a ions as in B. The elec o-
s a ic po en ial su aces we e c ea ed wi h a colo amp o posi i e (blue) and
nega i e ( ed) po en ials a 300 mMionic s eng h. The po en ials we e calcu-
la ed in Chime a so wa e (41).
MSP1
19
-Rus icyanin Complex
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