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 .