p. falciparum pfi1780w residues 85247 or 98247 , pfd1170c residues 132309 , and mal8p1.163 residues 131284 were cloned in vector pgex-6p-2 ( ge healthcare life sciences , glattbrugg , switzerland ) , and transformed into escherichia coli bl21(de3 ) .
pfe1605w residues 122335 and mal8p1.4 residues 310456 were cloned in popinf vector ( oxford protein production facility , harwell , uk ) , and transformed into e. coli rosetta2placi .
recombinant mal8p1.4 for antibody production was derived from a full - length codon - optimized gene cloned into pscodon ( eurogentec , seraing , belgium ) and expressed in the cherrycodon system ( eurogentec ) .
cells were grown in luria - bertani medium , supplemented with 1% ( w / v ) glucose for pfe1605w and mal8p1.4 . for nuclear magnetic resonance ( nmr ) samples , cells were grown in m9 medium supplemented with nh4cl and c6 
 d - glucose , and after protein induction were grown for 16 h at 18c .
cells were resuspended in phosphate - buffered saline ( pbs ; 150 mm nacl and 20 mm na2hpo4 , ph 7.4 ) and lysed by sonication ; lysates were spun at 24,000 g for 30 min .
lysate supernatants of phist domains cloned in pgex-6p-2 were incubated with glutathione sepharose resin ( ge healthcare lifesciences ) equilibrated in pbs , and proteins were eluted in a 50 mm tris - cl ( ph 7.8 ) , 12 mm reduced glutathione buffer .
the glutathione s - transferase ( gst ) tag was removed by 3c protease cleavage , followed by buffer exchange to pbs using a sephadex g-20 column ( ge healthcare lifesciences ) .
lysate supernatants of popnif - cloned phist domains were applied to a talon hitrap column ( ge healthcare lifesciences ) equilibrated in 20 mm na2hpo4 ( ph 7.4 ) and 300 mm nacl and eluted with a gradient to an imidazole - containing buffer ( 20 mm na2hpo4 , ph 7.4 ; 300 mm nacl ; and 500 mm imidazole ) . for pfe1605w ,
the his6 tag was removed by 3c protease cleavage during dialysis in 50 mm tris - cl ( ph 7.5 ) , 150 mm nacl , and 2 mm dithiothreitol ( dtt ) , followed by dialysis in 20 mm 2-(n - morpholino)ethanesulfonic acid ( mes ; ph 6.0 ) , 50 mm nacl , and 2 mm dtt .
pfe1605w was then applied to an sp ion exchange column ( ge healthcare lifesciences ) equilibrated in the dialysis buffer and eluted with a nacl gradient ( 20 mm mes , ph 6.0 ; 1 m nacl , and 2 mm dtt ) . for mal8p1.4 ,
the his6 tag was removed by 3c protease cleavage , followed by dialysis in 20 mm na2hpo4 ( ph 7.0 ) , 150 mm nacl , and 2 mm dtt .
final purification of all phist domains was performed by size - exclusion chromatography over a superdex s75 column ( ge healthcare lifesciences ) equilibrated in pbs supplemented with 1 mm dtt or buffer a ( 50 mm nacl ; 20 mm na2hpo4 , ph 7.0 ; and 1 mm dtt ) .
the cloning and protein production of pfemp1 intracellular segments , both full - length segments and fragments , and fluorescent labeling of the pf08_0141 ats were described earlier ( 32 ) .
a further 5 fluorescent - labeled full - length ats variants were produced in an analogous manner by substituting single amino acids for cysteines ; these are pff0010 ( g156c ) , pfb1055c ( q161c ) , pfc1120c ( q182c ) , pf08_0103 ( h154c ) , and pff0845c ( h159c ) .
protein identity was confirmed by matrix - assisted laser desorption ionization - time of flight mass spectrometry . unless otherwise noted , all biophysical experiments were performed in buffer a , except for analytical ultracentrifugation ( auc ) experiments in which 1 mm tris(2-carboxyethyl)phosphine was used instead of dtt .
fluorescence polarization measurements were recorded at 20c with a 5-fam label using a pherastar fs fluorimeter ( ex=485 nm , em=520 nm ; bmg labtech , ortenberg , germany ) .
differences in fluorescence polarization were fit using a single binding model in the program origin ( originlab , northampton , ma , usa ) .
auc velocity experiments were performed on 25 m protein samples using an optima xl - i analytical ultracentrifuge ( beckman coulter , fullerton , ca , usa ) .
sedimentation velocities were recorded by measuring absorbance at 280 nm , with 200 scans every 4 min at 10c and 35,000 rpm .
data were processed using sedphit ( 33 ) . the protein partial specific volume was calculated from the amino acid sequence .
a mosquito robot ( ttp labtech , melbourn , uk ) was used to set up 200-nl sized drops with a 1:1 ratio of protein to mother liquor .
pfi1780w residues 85247 at a concentration of 4.0 mg / ml were mixed with 0.1 m sodium acetate ( ph 4.6 ) and 2.0 m nacl buffer .
crystals developed in 7 d were cryoprotected by a brief incubation in mother liquor supplemented with 22.5% ( v / v ) glycerol , flash - cooled in liquid nitrogen , and diffracted up to 2.35  at the diamond light source ( harwell , uk ) , beamline i04 .
the space group was determined to be p3121 with 2 molecules / asymmetric unit . for phasing experiments
, the crystals were incubated with 250 mm 5-amino-2,4,6-triiodoisophthalic acid ( hampton research , aliso viejo , ca , usa ) for 5 min before cooling .
crystallographic data were integrated in xds ( 34 ) and scaled in scala ( 35 ) .
phase information for pfi1780w was obtained from a 2.44- resolution data set collected at a wavelength of 1.6531  using the diamond light source , beamline i04 . phasing by single - wavelength anomalous diffraction
was performed using phenix.autosol ( 36 ) , which located and refined 27 iodine atoms to produce a density map with initial figure of merit of 0.51 .
initial model building was done with phenix.autosol ( 247 residues built and 184 identified ) .
iterative model building was performed with coot ( 37 ) and refinement against the native 2.35  data was performed with buster 2.10 ( 38 ) .
the model and associated data have been deposited in the research collaboratory for structural bioinformatics ( rcsb ) protein data bank ( http://www.rcsb.org ) under accession number 4jle .
cs - rosetta ( 41 ) structure prediction was performed on residues 306346 of atss using backbone n , hn , and c chemical shifts recorded in the presence of the pfi1780w phist domain and extrapolated to complex saturation .
data were processed by talos+ ( 42 ) before input in cs - rosetta for fragment selection and generation of 12,800 models .
model superposition with a 3- root mean square deviation ( rmsd ) cutoff yielded large clusters of which the top 3 had 1313 , 943 , and 719 members .
these clusters showed characteristic -sheet structures but incomplete convergence due to flexible loops between  strands .
superposition using the most stable secondary structure elements ( residues 311314 , 320324 , and 329333 ) and 1- rmsd cutoff resulted in model convergence as judged by the funnel - shaped plot of model score vs. pairwise rmsd .
assignments of pfi1780w were performed at 37c using triple - resonance experiments on a 600-mhz avance iii spectrometer ( bruker , newark , de , usa ) with a cryogenic probehead .
nmr samples for assignments consisted of 0.2 mm c / n - enriched pfi1780w residues 98247 in buffer a ( ph 6.5 ) supplemented with 0.1 mm 4,4-dimethyl-4-silapentane-1-sulfonic acid , 0.02% ( w / v ) nan3 , and 5% ( v / v ) d2o .
the limited sample concentration and stability under these conditions ( typical lifetime of 2 d ) necessitated the use of multiple samples to obtain a sufficient signal / noise ratio .
assignments have been deposited in biomagresbank ( university of wisconsin , madison , wi , usa ; http://www.bmrb.wisc.edu/ ) under accession number 19719 .
perturbations in h and n chemical shifts were combined as (h , n ) using the formula 
 (h1 ,  n15)={[(h1)complex(h1)free]2 + 0.04  * [ (n15)complex(n15)free]2}1/2 similar perturbations of carbonyl c resonances were expressed as (c ) = (c)free  (c)complex .
full - length pfi1780w and pfe1605w inserts were c - terminally fused either to green fluorescent protein ( gfp ) into parl1a - gfp ( kindly provided by t. spielmann , bernhard nocht institut , hamburg , germany ; ref .
43 ) or c - terminally to hemagglutinin ( ha ) into pbcamr_3xha ( 44 ) .
falciparum strain 3d7 was cultured in human 0 + erythrocytes according to standard procedures ( 45 ) .
transfected parasites were drug selected with either 10 nm wr99210 ( jacobs pharmaceuticals , cologne , germany ) or 2.5 mg / ml blasticidin ( life technologies , zug , switzerland ) .
parasite proteins were obtained through saponin lysis of synchronized parasites ( 510% parasitemia ) after 2 sorbitol treatments within 4 h and percoll purified after 30 h. parasite aliquots were taken every 8 h. samples were run on a 12.5% ( w / v ) sds - page column with complete protease inhibitor cocktail ( roche , rozkreuz , switzerland ) and transferred to a nitrocellulose membrane ( hybond - c extra ; ge healthcare lifesciences ) .
antibodies were diluted in 5% ( v / v ) milk - pbs : mouse monoclonal anti - gfp ( 1:1000 ; roche ) , rabbit anti - ha ( 1:20 ; invitrogen , zug , switzerland ) , rabbit anti - mahrp1 ( 1:5000 ) , rabbit anti - mahrp2 ( 1:1000 ) , mouse monoclonal anti - glyceraldehyde-3-phosphate dehydrogenase ( gapdh ; 1:20,000 ) , mouse anti - pfe1605w ( 1:500 ) , mouse anti - pfi1780w ( 1:500 ) , and mouse anti - mal8p1.4 ( 1:500 ) .
binding was made visible by chemiluminescence ( supersignal west pico , thermo scientific , reinach , switzerland ) .
blood smears of infected parasite cultures were fixed in 100% acetone for 30 min ( 47 ) and blocked with 3% ( v / w ) bovine serum albumin .
primary antibodies , rabbit anti - mahrp1 ( 1:500 ) , mouse anti - pfi1780w ( 1:100 ) , mouse anti - pfe1605w ( 1:100 ) , mouse anti - mal8p1.4 ( 1:200 ) , mouse anti - gfp ( 1:100 , roche ) , and mouse anti - ats ( 1:50 ) , were incubated for 1 h ( 48 ) .
secondary antibodies ( goat anti - mouse alexa 488 , goat anti - mouse alexa 594 , and goat anti - rabbit alexa 594 ; invitrogen ) were incubated with 1 g / ml 4,6-diamidino-2-phenylindole ( dapi ; roche ) for 1 h at 1:200 dilution . alternatively , irbcs were fixed with 4% paraformaldehyde ( pfa)-0.01% glutaraldehyde and permeabilized with 0.1% triton x-100 .
slides were viewed with a zeiss lsm 700 confocal microscope ( carl zeiss gmbh , jena , germany ) , with a 63 oil - immersion lens ( 1.4 numerical aperture ) .
live parasites were imaged with a leica dm 5000b fluorescence microscope using a 100 oil immersion lens ( 1.4 numerical aperture ) with an attached leica dfc300fx camera and leica application suite software ( leica microsystems , heerbrugg , switzerland ) .
pfe1605w - ha and pfi1780w - gfp transfected mature parasites were purified by percoll density gradient , fixed in 2% pfa-0.2% glutaraldehyde in phosphate buffer , and prepared according to tokuyasu ( 50 ) .
ultrathin sections ( 7090 nm ) prepared on an fc7/uc7-ultramicrotome ( leica microsystems ) at 120c were immunogold - labeled with rabbit anti - ha ( 1:20 ; invitrogen ) or rabbit anti - gfp ( 1:20 ; abcam , cambridge , uk ) antibodies and 5 nm protein a - gold ( 1:70 ; umc , utrecht , the netherlands ) .
sections were stained with 4% uranyl acetate - methylcellulose ( 1:9 ) and examined with a transmission electron microscope ( cm10 or cm100 ; philips , eindhoven , the netherlands ) at 80 kv .
previously , we showed that intracellular segments ( referred to as atss ) from members of the pfemp1 family comprise a stably folded core and 3 flexible regions ( 32 ) .
we demonstrated that the phistc protein pfi1780w interacts with moderate strength ( kd150 m ) with the ats of pfemp1 variant pf08_0141 .
thus , we produced phist domains from proteins that had been reported as important for cytoadherence of irbcs ( pfd1170c ; ref .
4 ) , that are adjacent on the genome to the locus of pfemp1 variant pf08_0141 ( mal8p1.163 ) , or , alternatively , that were identified in proteomic studies on tethers ( pfe1605w ) and tested them for interactions with the ats . as a negative control
, we produced a phist member that was shown not to localize to the irbc membrane and thus would not be expected to associate with the ats ( mal8p1.4 ) .
polarization experiments showed that pfe1605w , a phistb member , bound fluorescently labeled ats pf08_0141 with 30-fold higher affinity ( kd=50.6 m ) than pfi1780w ( fig .
nmr experiments suggest that the phist interaction occurs at the c terminus of the ats ( see below ) , and polarization experiments using a fluorescently labeled ats c - terminal construct ( residues 293392 ) showed that this fragment is sufficient for strong pfe1605w binding ( fig .
b , c ) similar titrations with different labeled pfemp1 intracellular domains and pfe1605w ( b ) or pfi1780w ( c ) . binding by pfemp1 variant pff0845c was weak and could not be fit . in panel b ,
d ) titrations of ats pf08_0141 full - length or c - terminal fragment ( residues 293392 ) with pfe1605w . to test
whether different pfemp1 members retain the pfe1605w interaction , we produced fluorescently labeled atss from pfemp1 variants pff0010 , pfb1055c , pfc1120c , pf08_0103 , and pff0845c , which were selected on the basis of sequence divergence ( 32 ) .
intriguingly , polarization experiments with pfe1605w showed up to 25-fold difference in affinities ( kd range , 2.767 m ; fig .
whether these differences reflect closer associations of pfe1605w with particular pfemp1 members in vivo remains to be studied ; however , time course experiments did suggest a closer association of pfe1605w with pfemp1 than pfi1780w during transport . to test whether these phist proteins are exported and to determine their subcellular localizations , they were c - terminally gfp tagged and episomally expressed in 3d7 parasites under the control of the crt promoter . the integrity of the gfp - fusion proteins was shown on western blots ( fig .
the full - length pfi1780w - gfp was exported to the irbc cytosol similarly to the previously reported gfp - tagged n terminus ( 19 ) , but full - length pfi1780w - gfp additionally revealed fluorescence at the periphery of irbcs ( fig .
2b ; top panel ) , suggesting a localization close or adjacent to the erythrocyte membrane ( fig . 2b
immunofluorescent 3-dimensional reconstructions of fixed irbcs with pfi1780w - gfp - expressing parasites showed focal fluorescence in parasite cytosol and uniform fluorescence around the biconcave rim of the irbc ( fig .
available gfp - tagged phist proteins only pfe1605w - gfp gave a similar fluorescent pattern except that the rim - like fluorescence was observed at discrete foci instead of a uniform signal ( fig .
2b , panels 4 and 5 ) , indicating that both pfi1780w and pfe1605w were transported close to the irbc membrane .
all extracts are derived from saponin - released parasites , and the origin is indicated on top .
decoration of the same western blot with gapdh as loading control is shown at bottom .
b ) live cell imaging of 3d7 parasites expressing pfi1780w - gfp and pfe1605w - gfp .
for panels 2 and 5 , the focal plane of the gfp signal was set on the surface of the irbc , and the dapi / differential interference contrast ( dic ) microscopy signal was kept on the previous focal plane .
merge image of gfp / dapi / dic channels representing stack 32 of 69 in total .
d ) western blot of protein fractions of solubility assays of 3d7 parasites expressing pfi1780w - gfp and pfe1605w - gfp .
lane 1 , soluble proteins ; lane 2 , peripheral membrane proteins ; lane 3 , triton x-100 extract ; lane 4 , insoluble pellet .
the soluble parasite protein gapdh was found as expected in the supernatant after hypotonic lysis , and mahrp1 , an integral maurer 's cleft membrane protein ( 51 ) , was detected in the triton x-100 supernatant .
both pfi1780w - gfp and pfe1605w - gfp lack a predicted transmembrane domain and were shown to be membrane - associated proteins by solubilization in sodium carbonate similar to mahrp2 , a membrane - associated protein localizing to maurer 's cleft tethers ( 46 ) .
this result was further confirmed by the fluorescent labeling of the irbc membrane of a ruptured schizont expressing pfi1780w - gfp ( fig .
gold labeling of pfi1780w - gfp was found in proximity to the irbc membrane but was clearly absent from knobs ( fig .
3c ) and pfe1605w - gfp were clearly localized in knobs , and in trophozoite stage parasites , pfe1605w - gfp was also frequently found in maurer 's clefts ( fig .
3d ) , suggesting that it localizes underneath the irbc membrane , whereas pfe1605w localizes to knobs , but both proteins seem to be transiently transported through the maurer 's clefts .
shown here are postembedding immunoelectron microscopy images of irbcs expressing pfi1780w - gfp ( a , b ) , pfe1605w - ha ( c ) , and pfe1605w - gfp ( d ) .
white arrows , 5 nm gold . knobs ( k ) and maurer 's clefts ( mc ) are labeled .
scale bars = 200 nm ( a ) ; 500 nm ( c ) ; 250 nm ( b , d ) .
specific antisera against pfi1780w ( aa 80280 ) recognized a single band of 45 kda in agreement with its predicted mass of 45.5 kda ( fig .
4a ) , and antisera against pfe1605w ( aa 122335 ) recognized a band of 55 kda and a faint band of 50 kda .
western blotting using samples from 6 time points of the intraerythrocytic cycle showed that both proteins were present throughout the cycle , which is in agreement with their transcription profile ( 52 , 53 ) .
a ) western blot with extracts from stage - specific parasites ( hpi indicated for each lane ) .
samples were collected at 8-h intervals and stained with antibodies recognizing pfe1605w ( b ) , pfi1780w ( c ) , or ats ( d ) and costained with dapi .
we performed ifas to visualize expression and export of pfe1605w / pfi1780w throughout the intraerythrocytic cycle using tightly synchronized parasites at 6 time points ( fig .
4b , c ) and compared the timing of export of pfemp1 using anti - ats antibodies ( fig .
pfe1605w was first observed at 08 h postinfection ( hpi ) in a  necklace of beads  pattern at the parasite surface ( fig .
4b ; top panel ) and in young trophozoites ( 1624 hpi ) most fluorescence was observed in the parasite with few fluorescent foci beyond the parasite 's confines .
after 2432 hpi , the number of fluorescent foci in the irbc cytosol increased , and faint fluorescence was visible at the irbc membrane .
schizont stage parasites showed faint fluorescent dots at the irbc membrane , similar to those seen with live cell images of pfe1605w - gfp ( fig .
bright fluorescent foci indicated that pfe1605w is exported via maurer 's clefts to the irbc membrane and knobs .
pfemp1 is known to display a necklace of beads pattern at the parasite surface at 811 hpi ( 54 ) , which was confirmed ( fig .
4b ) . both proteins transiently associated with maurer 's clefts at 1624 hpi ( ref .
4b , d ) before being transferred to the irbc membrane , suggesting cotransport of pfe1605w with pfemp1 .
in contrast to pfe1605w , pfi1780w was found within the parasite cytosol until 2432 hpi with limited focal fluorescence in the irbc cytosol ( fig .
pfi1780w was exported to the irbc surface as shown in live cell imaging ( fig .
2b ) with no distinct intermediate locations , although a few fluorescent foci and immunoelectron microscopy data ( fig .
3b ) suggest that maurer 's clefts are intermediate transport compartments . to confirm the transient location of pfe1605w at maurer 's clefts
, we colocalized the protein with mahrp1 in ring , trophozoite , and schizont stage parasites .
while in ring stages , mahrp1 already appeared in maurer 's clefts and pfe1605w exclusively localized within the parasite cytosol ( supplemental fig .
pfe1605w and mahrp1 colocalized to maurer 's clefts with a partial overlap of signals , suggesting subdomains in maurer 's clefts as described previously for mahrp1 and pfemp1 ( 54 ) .
this is also evidence for pfe1605w transport to already formed maurer 's clefts and proof that cargo arrives at clefts independent of their formation as reported previously ( 54 , 55 ) . at later stages , pfe1605w dissociates from maurer 's clefts and
in contrast to pfe1605w and pfi1780w , mal8p1.4 , which does not interact with the ats domain ( fig .
the pfe1605w phist domain evaded crystallization alone or in complex with ats fragments and was unsuitable for extensive nmr analysis due to limited solubility .
thus , we turned to pfi1780w , which also interacts with ats , to gain information on the phist - ats complex . to delineate the specific phist - ats interaction site
, we performed nmr titrations of c / n - enriched ats pf08_0141 ( 32 ) with the unenriched pfi1780w phist domain ( residues 98247 ) .
5ac , the largest chemical shift perturbations , indicative of complex formation , span a wide region at the flexible c terminus of the ats .
although sequence similarity across the c terminus of pfemp1 intracellular segments is relatively low ( 32 ) , 75% of the amino acids most perturbed on pfi1780w binding are conserved ( i311 , i313 , i330 , l331 , d336 , i338 , y339 , y340 , and w391 ) or conservatively substituted ( t329 , d332 , e335 , i372 , and v390 ) in the majority of members of the pfemp1 family ( supplemental fig .
we were able to perform similar experiments on c / n - enriched ats with unenriched pfe1605w under dilute conditions and recorded the loss of ats resonance intensity in the presence of pfe1605w ( supplemental fig .
s3a ) . mapping interaction interfaces using this type of information is less sequence specific than that with the chemical shift perturbations observed with pfi1780w , as loss of resonance intensity may reflect interactions by a specific residue or its neighbors .
s3b ) suggest that pfe1605w affects a wide span of the ats c terminus that includes the pfi1780w interaction epitope ( fig .
in addition , pfe1605w makes additional contacts with the ats c terminus that span residues 303309 and 353362 ; these additional contacts may contribute to the higher ats affinity of this phist variant .
a ) combined perturbations of backbone amide nuclei of 0.1 mm c / n - enriched ats on titration with 0.2 mm unenriched pfi1780w phist domain .
dashed line denotes the level of average perturbation plus 1 sd ; residues with perturbations above this line are considered as significantly affected .
c ) carbonyl perturbations of the same ats region as in panel b. residues judged as significantly affected in either amide or carbonyl perturbations are colored based on their conservation : red if identical or orange if conservatively substituted in 75% of pfemp1 members in p. falciparum isolate 3d7 .
d ) n - heteronuclear single quantum coherence spectra overlay from 0.1 mm n - enriched pfi1780w phist domain alone ( red ) or in the presence of 0.2 mm unenriched ats c - terminal fragment ( residues 309392 , green ) .
e ) expansion of overlaid spectra from 50 m pfi1780w phist domain alone ( red ) , in the presence of 50 m ats residues 309392 ( black ) , after addition of 0.6 stoichiometric ratio of unenriched pfe1605w phist domain ( blue ) , or after addition of 1.2 stoichiometric ratio of pfe1605w ( green ) .
pfe1605w addition reverses pfi1780w perturbations caused by ats binding , suggesting that the 2 phist proteins compete for the same ats binding site .
f ) per - residue combined perturbations of backbone amide h and n nuclei from the pfi1780w spectra shown in panel d. dashed line identifies significantly perturbed residues as in panel b. to confirm that the 2 phist proteins compete for the same ats interaction epitope , we performed nmr experiments using n - enriched pfi1780w and unenriched ats c terminus and pfe1605w ( fig .
5d ) . perturbations on pfi1780w resonances caused by ats are reversed by pfe1605w ( fig .
we obtained crystals of a pfi1780w fragment spanning residues 85247 and solved the resulting structure to 2.35- resolution ( supplemental table s1 ) .
the final model showed 2 protein chains forming a tight helix - swapped dimer in the crystal ( fig .
6a ) ; however , auc velocity experiments showed that pfi1780w is monomeric in solution under physiological conditions ( fig .
thus , we concluded that the dimer is a crystallization artifact and consider only the monomeric unit of the phist domain .
a ) crystallographic model of the pfi1780w phist domain , showing the dimer ( blue and red chains ) .
data analysis yielded a narrow distribution at 1.65 s , with best friction ratio of 1.61 and 19,919 da corresponding molecular mass .
d ) twist of the triple - helix bundle for pfi1780w and spectrin ( rcsb 3kbt ) .
6c ) , in agreement with structure predictions ( 19 ) and circular dichroism data ( 32 ) . a short initial helix ( 1 )
is followed by an antiparallel bundle of 3 long helices ( 24 ) , which are up to 51 aa in length .
the pfi1780w triple - helical bundle is of length ( 6.5 nm ) comparable to that of a spectrin repeat ; however , in contrast to spectrin , it adopts a right - handed twist ( fig .
phist domains show remarkable sequence divergence , and pfi1780w belongs to the most variable phistc subtype of this family ( 19 ) .
alignment of the pfi1780w phist domain with its 5 closest relatives showed just 4% identical and 20.7% conservatively substituted residues ( supplemental fig .
the vast majority of these conserved amino acids , including all tryptophans characteristic of the phist family ( 19 ) , are involved in the protein hydrophobic core and do not form a continuous surface area . nonetheless , the pfi1780w structural model is sufficient to model with 98100% confidence the domain structures for all members of this family using automated prediction pipelines .
the chemical shift perturbations of pfi1780w on ats binding allowed us to map the binding site on the phist structure ( fig .
5f ) . the most significant changes localized at the middle of the pfi1780w triple - helical bundle , and , in particular , over a continuous surface on helices 2 and 3 ( fig . 7c and supplemental fig .
the amino acids forming this ats - binding interface ( k136 , e138 , e139 , h167 , f168 , and q171 ) are not conserved between pfi1780w and pfe1605w , which probably contributes to the different ats affinities of these proteins . nonetheless ,
because pfi1780w and pfe1605w compete for the same ats epitope , we reasoned that an analysis of the pfi1780w - ats interaction may provide a partial model of the high - affinity complex .
structure prediction for the ats c terminus and analysis of the pfi1780w - ats interaction .
models were predicted for a c - terminal fragment of ats spanning residues 306346 , using chemical shift restraints that correspond to the pfi1780w - complexed state of ats .
a characteristic funnel shape in this plot suggests that the calculation converged to a single subset of solutions .
b ) schematic representation of a low - energy ats model from panel a. c ) opposite views of the pfi1780w phist domain surface , with areas significantly affected by ats binding in nmr titrations shown in red .
d ) proposed mode of pfi1780w - ats binding based on the predicted structure of ats residues 306346 .
the number of ats - binding residues on pfi1780w is remarkably small compared with the broad span of perturbations observed on the ats c terminus ( fig .
, the continuous surface identified on pfi1780w 23 can only accommodate approximately 6 aa in an extended conformation . to understand how these observations relate , we predicted the transient structure adopted by the ats c - terminal epitope ( residues 306346 ) on pfi1780w binding using cs - rosetta ( 41 ) .
7a , b , the structure prediction converged to a single  sheet formed by ats residues 311314 ( 1 ) , 320324 ( 2 ) , 328334 ( 3 ) , and 337343 ( 4 ) .
formation of a single  sheet by ats residues 306346 may explain the widespread chemical shift changes seen in this construct on pfi1780w binding , as nearly all residues would transit from a random coil to an extended conformation . yet , at the same time , the single ats  sheet could provide a relatively narrow interaction interface with pfi1780w along the -sheet edge .
thus , we propose that an ats associates along one -sheet edge with pfi1780w ( fig .
the extensive remodeling of host erythrocytes by invading plasmodia is an impressive feat of biological engineering necessary for the parasites to grow , replicate , and evade the immune system ; thus , it has clear implications for the human host .
the unique alterations induced by p. falciparum resulting in irbc cytoadherence are linked to disease severity ( 7 ) . yet
although irbc remodeling involves hundreds of proteins , few interactions and even fewer structures have been examined in detail . here , we present the first structural - functional insights on phist proteins , which comprise a large subset of the p. falciparum exportome ( 4 , 19 ) . notably
, our study suggests strong links between a specific phist member and pfemp1 , the parasite receptor responsible for cytoadherence .
6 ) , are characterized by a relatively simple -helical bundle structure , a type of protein fold that can accommodate divergent sequences and , thus , present variable surface residues to accommodate different binding requirements .
the phist family in plasmodia probably serves precisely the role of a simple , easy - to - manipulate type of protein that can act to bridge or structurally support other components .
phist proteins seem to have a variety of functions in the parasite ( 4 , 20 , 21 , 26 , 30 ) , but this might reflect their role as interaction hubs that can bind flexible protein segments , as shown here for ats ( fig . 7 ) .
thus far , the molecular evidence of phist - mediated interactions presented here and elsewhere ( 20 , 21 , 56 ) involves only 10% of all family members , and high - throughput molecular methods may provide a clearer picture of the phist interactome .
what is the relationship of phist proteins with pfemp1 ? in a limited set of phist variants , we identified 2 members , pfi1780w and pfe1605w , that bound the pfemp1 intracellular segment ( fig .
1a ) ; it is likely that additional pfemp1-binding phist domains can be found among the 72 members of this family .
both phist proteins described here interact with the same pfemp1 epitope but with significantly different affinities .
furthermore , substantial variation in binding strength was observed , depending on specific phist / pfemp1 combinations ( fig .
1b ) , which leads us to propose that individual phist variants might have been optimized for different groups of pfemp1 molecules .
this proposal is strongly supported by transcriptomic evidence of a specific phista member ( pf14_0472 ) up - regulated in parasites expressing pfemp1 variants binding human brain endothelial cells ( 26 ) .
similarly , we observed that both pfemp1-binding phist proteins localized to the irbc membrane ( fig .
2b ) , but only pfe1605w was coexported with pfemp1 and localized in knobs ( fig .
( 56 ) showed that disruption of pfe1605w , termed lymp in their study , reduced irbc cytoadherence to cd36 by 55% , while retaining knob formation and pfemp1 surface localization .
further , they identified an interaction between inside - out vesicles prepared from uninfected human erythrocytes and the pfe1605w positively charged c terminus , but not its phist domain . together with the data presented here
, we can now suggest at least one mechanistic role for pfe1605w and potentially for other phist proteins in cytoadherence ( fig .
1 ) soon after export from the parasite and remains bound during trafficking to maurer 's clefts ( fig .
3 ) . when in knobs , the c - terminal segment of pfe1605w links pfemp1 with the host cytoskeleton .
7 ) probably complements direct ats - cytoskeletal binding ( 57 ) and ensures mechanical robustness against shear forces exerted by blood flow , thereby allowing strong cytoadherence .
our proposed mechanism of pfe1605w function , connecting the c terminus of pfemp1 ats to the cytoskeleton , is illustrated .
in summary , we present here the first structural insights on members of the phist family and how they function as protein interaction modules .
the interaction properties of specific members could be linked to their cellular localization and trafficking pattern alongside the well characterized pfemp1 , allowing the proposition of a mechanistic model of the function of phist molecules in cytoadherence .
 uniquely among malaria parasites , plasmodium falciparum - infected erythrocytes ( irbcs ) develop membrane protrusions , known as knobs , where the parasite adhesion receptor p. falciparum erythrocyte membrane protein 1 ( pfemp1 ) clusters . 
 knob formation and the associated irbc adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the irbc . 
 a family of exported proteins featuring plasmodium helical interspersed subtelomeric ( phist ) domains has attracted attention , with members being implicated in host - parasite protein interactions and differentially regulated in severe disease and among parasite isolates . here 
 , we show that phist member pfe1605w binds the pfemp1 intracellular segment directly with kd = 5  0.6 m , comigrates with pfemp1 during export , and locates in knobs . 
 phist variants that do not locate in knobs ( mal8p1.4 ) or bind pfemp1 30 times more weakly ( pfi1780w ) used as controls did not display the same pattern . 
 we resolved the first crystallographic structure of a phist protein and derived a partial model of the phist - pfemp1 interaction from nuclear magnetic resonance . 
 we propose that pfe1605w reinforces the pfemp1-cytoskeletal connection in knobs and discuss the possible role of phist proteins as interaction hubs in the parasite exportome.oberli , a. , slater , l. m. , cutts , e. , brand , f. , mundwiler - pachlatko , e. , rusch , s. , masik , m. f. g. , erat , m. c. , beck , h .- 
 p . 
 , vakonakis , i. a plasmodium falciparum phist protein binds the virulence factor pfemp1 and comigrates to knobs on the host cell surface . 
MATERIALS AND METHODS
RESULTS
DISCUSSION