bacterial toxins of the cholesterol - dependent cytolsysin ( cdc ) superfamily form pores in lipid bilayers through the oligomerization of their membrane attack complex / perforin fold ( macpf ) domain .
cdcs are produced by over five different genera of gram - positive bacteria , resulting in a wide range of animal and human diseases ( tweten et  al . , 2001 ) .
intermedilysin ( ily ) is a cdc secreted by streptococcus intermedius that causes brain and liver abscesses in its host ( nagamune et  al . , 1996 ) and is thought to be the major virulence factor for the bacterium ( nagamune et  al . , 1996 ) .
cholesterol is essential for ily cytolytic activity , and crystal structures of ily and other cdcs show strong structural homology ( bourdeau et  al . , 2009 ; polekhina et  al . , 2005 ;
rossjohn et  al . , 1997 ; rossjohn et  al . , 2007 ; xu et  al . , 2010 )
most cdcs bind to cholesterol - rich membranes via a cholesterol recognition motif ( crm ) and neighboring undecapeptide .
this binding triggers the oligomerization and allosteric regulation of the transition to the pore state ( dowd and tweten , 2012 ) .
ily , on the other hand , has evolved to recruit the human complement regulator cd59 as a receptor providing the basis for its species specificity ( giddings et  al . , 2004 ) .
it is cd59-binding that promotes both oligomerization of ily monomers on the cell surface ( lachapelle et  al . ,
2009 ) and the structural rearrangements required for pore formation ( dowd and tweten , 2012 ) . in contrast , the interaction of cholesterol with the crm and undecapeptide is required for the maintenance of the ily prepore at the membrane as cd59 is released ( lachapelle et  al . ,
mutational analysis of ily has determined residues involved in both lipid and receptor binding ( wickham et  al . , 2011 ) as well as those crucial for mediating structural transitions ( dowd and tweten , 2012 ; hotze et  al . , 2012 ; lachapelle et  al . , 2009 ;
our structure of the ily - cd59 complex now provides a framework with which to integrate these biochemical and biophysical data .
cd59 is the only membrane - bound inhibitor of the complement terminal pathway ( meri et  al . ,
this small ( 77 amino acid ) gpi - anchored cell - surface receptor binds complement components c8 and c9 ( ninomiya and sims , 1992 ) and directly inhibits the formation of the membrane attack complex ( mac ) , an immune pore that kills microbes by the disruption of lipid bilayers .
cd59 is expressed on a variety of cell types , including leukocytes , epithelial , and endothelial cells ( meri et  al .
, 1991 ) and plays an important role in preventing complement - mediated tissue damage in hosts .
cd59 has been implicated in a number of human diseases , including tumor cell evasion during monoclonal antibody treatments ( fishelson et  al . , 2003 ; gorter and meri , 1999 ) and paroxysmal nocturnal hemoglobinuria ( hill et  al . , 2006 ) .
structural studies of soluble cd59 ( fletcher et  al . , 1994 ; kieffer et  al . ,
1994 ; leath et  al . , 2007 ) and direct binding assays of cd59 with complement components ( huang et  al . ,
2006 ) have provided some insight into the role of this receptor in regulating mac assembly . however , a lack of structural information characterizing cd59 with its binding partners has limited our ability to control complement susceptibility . here , we report the crystal structure of cd59 bound to ily , a bacterial toxin that directly competes for mac binding .
we define two interfaces on cd59 that coordinate ily monomers and validate this interaction through the development of a liposome - based model system .
we also find that an ily - derived peptide based on our structure disrupts the interface and competes for pore formation .
in addition , cryo - electron microscopy ( cryo - em ) images of the crystallized ily mutant bound to cd59-decorated liposomes suggest that the linear arrays observed in our crystal could also occur in the context of a membrane .
altogether , our structural and functional data provide mechanistic insight into how cd59 engages ily for the formation of a pore in lipid bilayers , providing a basis for the development of new antibiotics as well as potential therapeutics that target cd59 .
an early prepore intermediate comprising the initial attachment of ily to cd59 was captured with a disulfide - locked ily mutant ( ily ) and a cd59 construct lacking the gpi anchor .
the complex was assembled in  vitro , subjected to further purification , and crystallized .
the crystals diffracted to a resolution of 3.5   , and the structure was solved by molecular replacement .
the final model , consisting of all four domains of ily and the  ectodomain of cd59 , was refined to a final rwork / rfree of  21.2%/26.6% and displayed good geometry ( figures 1 and  s1a ; table 1 ) .
as described previously , ily is comprised of four structural domains , the first three of which are formed from discontinuous stretches of sequence .
domain 1 ( d1 ) ( residues 5676 , 117201 , 258299 , and 379399 ) forms a cap at the top of the molecule and is the least well - ordered region of our crystal ( figure  s1b ) .
domain 2 ( d2 ) ( residues 77116 and 400417 ) consists of three kinked  strands that span 50   and bridge domains 1 and 4 .
domain 3 ( d3 ) ( residues 202257 and 300378 ) packs against d2 and contains the helical bundles that are predicted to refold into transmembrane -hairpins .
this domain , along with d1 , makes up the macpf domain and also contains the site of the engineered disulfide bond ( c346-c361 ) that prevents the structural rearrangements required for full pore formation . finally , domain 4 ( d4 )
( residues 418528 ) is a discrete  sandwich structure extending from the end of d2 .
cd59 is a compact , heavily disulfide - bonded structure with a central  sheet bookended by a short  helix and a two - strand  hairpin .
the asymmetric unit of the crystal contains two copies of the ily - cd59 complex , which overlay with an rmsd of 1.0   over 530 residues .
cd59 predominantly interacts with d4 of ily ( figure  1a ) , which is in agreement with previous studies ( nagamune et  al .
2005 ) . strikingly , there are two major ily - cd59 interfaces within the crystal : one within the asymmetric unit and one between neighboring unit cells ( figure  1b ) . the primary ily - cd59 binding site ( 665   buried surface area )
is composed of a side - to - side contact between the  hairpin extension of ily d4 ( the 2-3 loop ) and the core  sheet of cd59 ( figure  1c ) , effectively creating an extension of the cd59 sheet .
the interface is further stabilized by the stacking of hydrophobic side - chains ( ily : y436 , ily : i450 , cd59:f47 , cd59:y61 , and cd59:f42 ) as well as an extensive network of hydrogen bonds ( both sidechain - sidechain and sidechain - backbone ) and  salt bridges ( ily : r480 , cd59:e58 ; ily:448 , cd59:k65 ; ily : d445 , and cd59:k66 ) .
this interface is supported by two independent mutagenesis studies ( hughes et  al . , 2009 ;
specifically , ily double ( y434a / y436a ) and triple ( s499n / k500r / n501 t ) mutants located within our primary cd59 interface ( figure  1c ) dramatically inhibited pore formation and decreased the affinity of ily for cd59 .
in addition , double- and single - point mutations in cd59 have implicated aromatic residues that map to this interface ( f42 , f47 , and y62 ) as being involved in ily binding ( wickham et  al . , 2011 ) .
the secondary binding site is formed by the same region of ily ( the 2-3 loop in d4 ) interacting with cd59 from a neighboring unit cell .
this interface buries 500   and is stabilized by a series of hydrogen bonds and three salt bridges ( ily : d443 , cd59:r55 ; ily : d445 , cd59:k38 ; ily : e93 , and cd59:k41 ) .
the second interface is again validated by previous mutagenesis studies of both ily and cd59 ( wickham et  al . , 2011 ) .
in particular , a double mutation in ily ( r451a / s452a ) that reduces cd59-binding and hemolysis maps to this region ( figure  1d ) .
cd59 mutations d22 and f23 , also located in this interface ( figure  1d ) , play a role in ily binding and function .
therefore , cd59 displays two distinct binding sites for ily that can be simultaneously occupied .
this suggests that , in addition to localizing ily to the membrane surface , cd59 helps nucleate an early prepore complex by bridging neighboring ily molecules , thereby creating the linear array observed in our crystals . to address the functional significance of the ily - cd59 interfaces observed in our crystal structure , we developed a simple model membrane system to probe ily pore formation in a fluorescence - based assay ( koer et  al . , 2007 ) .
calcein - containing liposomes were decorated with soluble cd59 and modified to possess a myristoylated lysine - rich  cytotopic  peptide ( fraser et  al . , 2003 ) .
lipid attachment of cytotopic cd59 was confirmed by the colocalization of protein in lipid - containing fractions across a ficoll gradient ( figure  2a ) .
the fluorescence intensity of calcein released from liposomes upon lysis was used to determine the rate of pore formation .
exposure of wild - type ily ( ily ) with cholesterol - containing cd59-decorated liposomes resulted in functional transmembrane pores ( figure  2b ) , whereas undecorated liposomes did not support lysis ( figure  2b ) . furthermore , incubation of liposomes with cytoptic cd59 alone did not leak calcein ( figure  2b ) . to confirm binding of the ily  hairpin extension to cd59
, a peptide comprised of ily residues 438452 was synthesized with bridging n- and c - terminal cysteines .
the peptide successfully competed for cd59 binding and inhibited ily pore formation with an ic50 of approximately 25  m ( figures 2c and s2 ) . to understand how cd59 coordinates ily monomers to form the pore
, we characterized the complex in a lipid environment using electron microscopy . because negatively stained specimens could lead to distortions of liposome curvature and possible liposome collapse , we used cryo - em to visualize membrane - bound complexes
. examination of cryo - em fields of ily - cd59-liposome complexes revealed a population of liposomes with multiple instances of embedded circular pores ( figure  3a ) .
density is clearly visible for the ily protein rings with obvious disruption to the lipid bilayer .
the diameter of the ily pore is approximately 300   , consistent with the dimensions previously reported for other cdc and perforin pores ( law et  al . , 2010 ; tilley et  al . ,
2005 ) . to test the ability of the crystallized ily mutant ( ily ) to form linear arrays in the context of a membrane , we examined two - dimensional cryo - em images of the mutant form bound to cd59-decorated liposomes ( figure  3b ) .
as expected , no perforation of the lipid bilayer was observed with this prepore intermediate .
protein density on the periphery of liposomes appears to be organized in linear clusters ; however , due to the fragility of the system , we were unable to collect in - plane images to confirm this .
reversible linear arrays of a prepore - trapped mutant form of perfringolysin have demonstrated that cdcs undergo transient , yet specific , intermolecular interactions that precede ring closure ( hotze et  al . , 2012 ) .
these data suggest that the linear organization of ily - cd59 complexes observed in our crystal structure is relevant for understanding how ily engages with cd59 in the context of a membrane .
previously reported cryo - em reconstructions of the pneumolysin prepore and pore structures have provided a model for how cdcs oligomerize on membranes ( tilley et  al . , 2005 ) .
preserving the two ily - cd59 interfaces observed in our crystal structure , we used this as a starting point to turn the linear array of our lattice into a model for the ily early prepore state ( see experimental procedures for details ) .
this arrangement places cd59 at the periphery of the ring ( figure  4 ) , and we find that the presence of cd59 would not obstruct ily oligomerization , in concordance with data showing that cd59 remains bound during prepore assembly ( lachapelle et  al . , 2009 ) .
these data , along with the high concentration of cd59 on the cell surface ( anstee , 1990 ) , have led us to include cd59 in our model of an ily prepore oligomeric ring .
the subsequent release of cd59 during the pore transition , observed previously by biophysical studies ( lachapelle et  al . , 2009 )
, could be due in part to conformational changes that are incompatible with maintaining cd59 in the complex . in our model
, the c - terminal residue of cd59 preceding the gpi anchor is positioned approximately 15   from the plane of the membrane ( figure  4 ) .
this observation is consistent with distances measured for other gpi - anchored proteins ( lehto and sharom , 2002 ) and with a model previously proposed for cd59 glycosylation ( rudd et  al . , 1997 ) .
therefore , our structural and functional data together suggest that the role of cd59 in ily pore formation is to orient the ily crm and undecapeptide proximal to cholesterol - containing lipid bilayers and to propagate ily monomer - monomer interactions through two binding interfaces .
polekhina et  al . , 2005 ; rossjohn et  al . , 1997 ; rossjohn et  al .
, 2007 ; xu et  al . , 2010 ) and components of the complement mac ( aleshin et  al . , 2012 ; hadders et  al . , 2007 ;
2011 ) reveal that the fundamental pore - forming mechanism of these macpf - containing proteins are similar .
however , within this conserved mechanism , one of the most important differences remains in its regulation . for many
cdcs , membrane binding by the crm and undecapeptide  initiates structural transitions ( dowd and tweten , 2012 ) , which are propagated between membrane - bound monomers ( hotze et  al . , 2012 ) .
circularization of the cdc oligomeric ring then triggers a dramatic conformational change in which helical segments from d3 convert to amphipathic hairpins and arrange into a transmembrane -barrel pore ( tilley et  al . , 2005 ) .
for a subclass of cdcs , of which ily is an archetypal member , control of regulating prepore transitions is transferred from the undecapeptide to cd59 binding ( dowd and tweten , 2012 ) .
our structural and functional analysis of the ily - cd59 complex supports a model for these pores in which membrane - anchored cd59 recruits soluble ily monomers to the plasma membrane of human cells , orienting the crm and undecapeptide for insertion into cholesterol - containing lipid bilayers . through two distinct ily - binding interfaces on opposite sides of the molecule
, cd59 brings together ily monomers in order to facilitate intermolecular interactions that drive the formation of an oligomeric prepore ring .
relatively small alterations to the packing of our crystal lattice produces a 31-fold symmetric model for this early prepore state that agrees with a pneumolysin prepore model ( tilley et  al . , 2005 ) .
even smaller manipulations of our lattice produce alternate models for ily pores in line with the proposed stoichiometries observed for other cdcs by different biophysical techniques ( 31- to 44-fold ) ( czajkowsky et  al . , 2004 ; tilley et  al . ,
monomer - monomer interactions of cdcs have been shown to propagate early structural transitions required for pore formation ( hotze et  al . , 2012 ) .
specifically , the positioning of neighboring molecules has been suggested to allow the intermolecular pairing of 1 from d3 of one molecule with 4 from the equivalent domain of the next .
analysis of the impact of applying curvature to our linear array shows that these contacts are indeed partially formed in our model ( movie s1 ) . in order to complete the zippering of the sheet - formation , 5 would need to swing out of the way .
this transition is prevented in our crystal by the engineered disulfide bond in ily locking 5 to 4 , thus trapping the earliest prepore state .
although ily and mac components ( c8 and c9 ) all bind cd59 , the result of this interaction has profoundly different outcomes on pore formation .
although cd59 promotes the oligomerization of ily monomers ( lachapelle et  al . , 2009 ) , the receptor inhibits mac formation by preventing membrane perforation and incorporation of multiple c9 molecules ( farkas et  al . ,
2002 ; meri et  al . , 1990 ; rollins and sims , 1990 ) .
ily directly competes for the mac binding site on cd59 ( lachapelle et  al .
, 2009 ; wickham et  al . , 2011 ) , and a mutational analysis has confirmed that the ily and mac binding sites on the surface of cd59 overlap , at least in part ( bodian et  al .
, 1997 ; huang et  al . , 2005 ; wickham et  al . , 2011 ) .
cd59-binding mac components lack the structural equivalent of ily d4 , therefore precluding further insight into how specific residues of c8 and c9 interact with the complement receptor .
however , it is possible that cd59 engages the transmembrane segments of complement components after their transition to  hairpins but prior to membrane insertion , similar to the interaction of cd59 with the  hairpin extension of ily d4 .
it is interesting to note that the residues on cd59 involved in mac binding map to the same two opposing faces observed in our ily interaction .
cd59 inhibits mac assembly most efficiently when incorporated prior to the addition of c9 ( lehto and meri , 1993 ) .
subsequently , it appears to lock a single c9 molecule into the complex in a conformation that prevents membrane perforation and further c9 recruitment ( lehto and meri , 1993 ; meri et  al . ,
these observations , along with the ily - cd59 crystal structure , suggest a mechanism for mac inhibition whereby cd59 intercalates between c8 and c9 in an analogous manner to the ily nucleation , locking the mac in a nonfunctional state . in summary ,
our findings provide mechanistic insights into how ily pores form , revealing a dual role of cd59 in facilitating the assembly of the oligomeric prepore .
our structural and functional data have defined two cd59-ily interaction interfaces , providing a framework for the development of new antibiotics and cd59-targeted therapeutics that could improve human health .
recombinant soluble human cd59 with an additional c - terminal cysteine residue was expressed in e.coli , refolded , and purified from solubilized inclusion bodies as described previously ( leath et  al . , 2007 ) .
purified cd59 ( approximately 0.5  mm in phosphate - buffered saline [ ph7.3 ] pbs ) was treated for 16  hr at ambient temperature with two molar equivalents of tris-2-carboxyethyl phosphine ( tcep , sigma - aldrich ) for the generation of a free thiol at the carboxyl terminal cysteine of the protein .
this was incubated for 2  hr at ambient temperature with a 3-fold molar excess of the cytotopic modification reagent bis - myristoyl lysyl sskkspskkddkkpgd ( s-2-thiopyridyl)-cysteine acid ( apt3146 , cambridge research biochemicals ) for the generation of cytotopic cd59 ( hill et  al . , 2006 ) .
the main protein peak fractions were pooled , buffer exchanged into pbs , and stored at 70c until use .
an ily gene lacking cysteine residues ( ily ) and a disulfide - locked ily mutant containing the substitutions t346c and i361c ( ily ) were cloned into a ptrchisa vector and were generous gifts from r. tweten .
this early prepore form of ily was characterized previously ( lachapelle et  al . , 2009 ) and shown to be converted to functional pores by the addition of reducing agent .
cells were grown to an optical density of 0.6 at 37c and induced with 0.5  mm iptg for 4  hr .
cultures were pelleted and lysed in a buffer containing 1% triton , 200  mm nacl , 20  mm tris ( ph 6.8 ) , protease inhibitor mixture ( sigma - aldrich , 50  l per gram of cell pellet ) , 100  g of dnase i , and 100  g of rnase a ( sigma - aldrich ) .
cleared lysates were loaded onto cobalt - chelated talon beads ( clontech ) and eluted with 500  mm imidazole .
ily was further purified by size exclusion chromatography in 150  mm nacl and 20  mm tris ( ph 6.8 ) .
ily and cd59 were incubated at room temperature in a 1:2 molar ratio , and the complex was separated on a superdex 200 hr 10/30 column ( pharmacia biotech ) .
crystals were grown by vapor diffusion at room temperature in 96-well plates with 0.4  l droplets at 21c .
equal volumes of preformed ily - cd59 complex ( 9.9  mg / ml in 150  mm nacl , and 20  mm tris [ ph 6.8 ] ) and precipitant ( 20% peg6000 , 0.1  m hepes [ ph 7 ] , and 0.2  m licl ) were mixed .
crystals were cryo - protected with reservoir solution plus 20% glycerol before cryo - cooling in liquid nitrogen .
data were collected at diamond light source ( harwell science and innovation ) at beamline i04 .
diffraction data were processed with xia2 ( winter , 2010 ) in 3diir mode and initially assigned as p21 with a single copy of the complex per asymmetric unit ( indexing initially suggested c222 ( 1 ) , but this failed upon scaling , see the twinning discussion below ) .
the structure of the ily - cd59 complex was solved by molecular replacement with the use of phaser ( mccoy et  al . , 2007 ) .
a solution was reached with the use of the known structures of ily ( pdb 1s3r ) ( polekhina et  al . , 2005 ) and cd59 ( pdb 2uwr ) ( leath et  al . , 2007 ) , with the ily split into two search models encompassing d1d3 and d4 .
. , 2010 ) suggested that the data were twinned , and the structure was refined with phenix ( adams et  al . , 2010 ) with the pseudomerohedral twinning operator ( h ,- k ,-
analysis of the resultant maps showed poor density for large stretches of d1 with no extra density or residuals , and the rfree did not drop below 0.30 .
therefore , we reprocessed the data in p1 ( xia2 , 3da mode ) and resolved the structure with two copies of the complex in the asymmetric unit .
analysis of the p1 data set with xtriage suggested twinning with only one of the three possible pseudomerohedral twinning operators ( h ,- k - l ) significantly occupied ( twin fraction  = 0.46 ) .
although the data merge well to 3.5   in both p21 ( rpim  = 0.077 , rmerge  = 0.105 ) and p1 ( rpim  = 0.098 , rmerge  = 0.111 ) , refinement carried out in the p1 data set in phenix with the h ,-
after rebuilding in coot ( emsley et  al . , 2010 ) , the rwork / rfree converged to 0.212/0.266 ( compared to 0.248/0.300 for the same model in the p21 data ) .
furthermore , the p1 maps showed clear features that differed between the two independent copies of the complex  in particular , the engineered disulfide ( cys346-cys361 ) was clearer in one copy  and the pattern of thermal motion factors over the two molecules reproducibly refined to different distributions .
some stretches of d1 still had weak or no electron density associated with the model , although this varied between the two copies .
however , refinement statistics worsen when these regions are removed , and molecular replacement of this domain placed it with z score > 15 in phaser . therefore , we decided to refine d1 in place and not rebuild in the density .
the final refined p1 model gave good molprobity statistics for the resolution ( molprobity score  = 2.28 , 99 percentile ) .
running xtriage with the final refined model in p1 gave r versus r statistics ( lebedev et  al .
l operator of 0.132/0.373 ( robs / rcalc ) , consistent with twinning with rotational pseudosymmetry . a model for an early prepore was generated from the crystal lattice by bending a linear array containing 31 copies of the 1:1 ily - cd59 complex into a circle . in brief , the linear array was placed onto the deposited pneumolysin prepore model ( pdb 2bk2 ) by the superposition of d1d3 with secondary structure mapping ( lsqkab ) .
then , a 15.3 rotation was applied to make the linear array tangential to the circular prepore density ( emdb 1106 ) .
the linear array was then bent into the circular form by the application of a 0 , 0 , 11.6 rotation ( polar angles ) to a monomer .
the cd59 n - linked glycan on asn18 and the gpi anchor were modeled with the coordinates described in rudd et  al . , 1997 , and kindly supplied by m. wormald .
1,2-dioleoyl - sn - glycero-3-phospho - l - serine ( dops ) , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine ( dope ) , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , and cholesterol ( supplied in chloroform from avanti polar lipids ) were mixed in molar ratios of 3:3:2:2 , respectively .
chloroform was evaporated with a stream of nitrogen gas , leaving a thin lipid film .
dried lipid was rehydrated with four freeze , thaw , and vortex cycles followed by extrusion through a 0.1  m pore - size membrane ( avanti ) for the formation of a homogeneous population of unilamellar liposomes , freshly prepared for each experiment .
fluorescently - labeled liposomes were generated by in the inclusion of 50  mm calcein ( sigma - aldrich ) in the rehydration buffer .
this initial calcein concentration of 50  mm is sufficient to cause almost complete self - quenching of its fluorescence ; if calcein is subsequently released from the liposomes , its concentration is reduced and an increase in fluorescence is observed .
unencapsulated dye was removed by passage through a g-50 sephadex column ( sigma - aldrich ) run in a buffer containing 500  mm sucrose .
lipids were tracked by the inclusion of phosphatidylethanolamine with lissamine rhodamine b - labeled head groups ( avanti ) in the lipid mixture described above at a final concentration of 2% ( w / w ) prior to the evaporation of chloroform .
mg / ml were incubated with either soluble or cytotopic cd59 at a final concentration of 18  m .
the liposome sample was adjusted to contain ficoll at a final concentration of 20% ( w / v ) and was overlaid with 10% ( w
after centrifugation at 100,000   g at 20c for 45  min , gradients were fractionated from the top .
fractions were analyzed for the presence of liposomes by the visualization of rhodamine and for the presence of cd59 by sds - page and coomassie staining .
pore - formation assays were performed at 20c in a varian cary eclipse fluorescence spectrophotometer ( agilent technologies ) with excitation and emission wavelengths of 490  nm and 520  nm , respectively , and a slit width of 5  nm .
the kinetics mode of the spectrophotometer was used with an average read time of 0.15 s , and measurements were made every minute .
a peptide corresponding to the cd59-binding interface of ily ( residues 438452 ) was synthesized with bridging n- and c - terminal cysteines ( ceelghdadgyetirsc ) ( severn biotech ) .
cytotopic cd59 at a final concentration of 0.7  m was incubated with 200  l of fluorescently - labeled liposomes in the presence or absence of peptide , and the average background fluorescence intensity was measured for 10  min . subsequently ,
ily at a final concentration of 0.7  m was added to the reaction and the fluorescence was measured for 45  min .
liposomes were burst at the end of the experiment by the addition of 1  l 0.2 m c12e8 detergent ( sigma - aldrich ) , and the maximum fluorescence was found by monitoring the sample for an additional 10  min .
the fluorescence measurements for each reaction were normalized according to the background and detergent readings .
rates of calcein release ( which corresponds to pore formation ) were calculated by fitting the normalized fluorescence intensity data with a nonlinear fitting algorithm in originpro 8.6 .
we incubated 0.75  mg ml liposomes with cytotopic cd59 and either ily or ily in equimolar ratios so that the final protein concentration was 1  mg / ml . we applied 2.5  l of the mixture to glow discharged holey carbon grids ( quantifoil r 2/2 ) and vitrified in liquid ethane with an fei vitrobot .
images were acquired on a cm200 feg electron microscope ( fei ) operating at 200 kv .
images were recorded with a defocus range of 2.5 to 3.5 microns underfocus at a magnification of 38,000 and an electron dose of 20 e /  with a 4k  4k temcam - f415mp ccd camera ( tietz video and image processing systems ) .
 summarypore - forming proteins containing the structurally conserved membrane attack complex / perforin fold play an important role in immunity and host - pathogen interactions . 
 intermedilysin ( ily ) is an archetypal member of a cholesterol - dependent cytolysin subclass that hijacks the complement receptor cd59 to make cytotoxic pores in human cells . 
 ily directly competes for the membrane attack complex binding site on cd59 , rendering cells susceptible to complement lysis . to understand how these bacterial pores form in lipid bilayers and the role cd59 plays in complement regulation , we determined the crystal structure of human cd59 bound to ily 
 . 
 here , we show the ily - cd59 complex at 3.5   resolution and identify two interfaces mediating this host - pathogen interaction . 
 an ily - derived peptide based on the binding site inhibits pore formation in a cd59-containing liposome model system . 
 these data provide insight into how cd59 coordinates ily monomers , nucleating an early prepore state , and suggest a potential mechanism of inhibition for the complement terminal pathway . 
Introduction
Results
Discussion
Experimental Procedures