Antimalarial activity of cupredoxins: the interaction of Plasmodium Merozoite Surface Protein 119 (MSP119) and Rusticyanin

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. 20896 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20897 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 20898 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20899 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 20900 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20901 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om (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 20902 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20903 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om 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 20904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 a FAC BIOLOGIA/BIBLIOTECA on Feb ua y 12, 2018h p://www.jbc.o g/Downloaded om