fusion of haploid cells to form a diploid zygote is the defining event of sexual reproduction in eukaryotes ( lillie , 1913 ) . in organisms from every eukaryotic taxon ,
the plasma membranes of gametes of opposite sex or mating type come into intimate contact and then fuse to form the zygote ( bianchi et  al .
very little is known about the molecular mechanisms of the membrane fusion reaction between gametes , and a bona fide fusion protein has not been formally identified . the best candidate to date is the ancient gamete plasma membrane protein hap2 , whose presence in green algae , higher plants , unicellular protozoa , cnidarians , hemichordates , and arthropods ( cole et  al . , 2014 , ebchuqin et  al . , 2014 , johnson et  al . , 2004 , kawai - toyooka et  al . , 2014 , liu et  al . , 2008 , mori et  al . , 2006 ,
steele and dana , 2009 ) indicates it was likely present in the last eukaryotic common ancestor ( leca ) ( wong and johnson , 2010 ) .
hap2 was first identified in a screen for male sterility in the flowering plant arabidopsis thaliana ( johnson et  al . , 2004 ) and later  under the name gcs1 ( mori et  al .
, 2006)as a sperm - specific protein shown to be required at an unidentified step in sperm - egg fusion ( mori et  al . , 2006 ,
a screen for genes essential for gamete fusion in  the green alga chlamydomonas independently uncovered hap2 , showing that it is expressed only in minus gametes and is exclusively present on an apically localized membrane protuberance termed the minus mating structure ( liu et  al .
, 2008 ) ( see figure  1a for a diagram of chlamydomonas fertilization ) .
studies in chlamydomonas and plasmodium ( the pathogen causing malaria in humans ) revealed that hap2 mutant gametes in both organisms were fully capable of robust adhesion to gametes of the opposite mating type or sex , but merger of the lipid bilayers was abrogated ( liu et  al . , 2008 ) . in both organisms , adhesion relies on proteins that are species - limited : fus1 in chlamydomonas plus gametes and its unidentified receptor in minus gametes ( misamore et  al . , 2003 ) , and p48/45 in plasmodium berghei gametes ( van dijk et  al . ,
2001 ) . based on these findings , which have since been confirmed in arabidopsis thaliana ( mori et  al . , 2014 ) and the ciliated protozoan tetrahymena thermophila ( cole et  al . , 2014 ) , a model emerged positing that hap2 , a single - pass transmembrane protein , functions after species - limited adhesion in the membrane fusion process between gametes ( liu et  al . , 2008 ) .
furthermore , in all of these organisms , hap2 is required in only one of the two gametes , raising the possibility that it may function similarly to fusion proteins of enveloped viruses ( wong and johnson , 2010 , harrison , 2015 ) . to understand the function of hap2 at the molecular level we carried out concerted bioinformatic , functional , and x - ray structural analyses of hap2 from chlamydomonas reinhardtii .
initial bioinformatic analyses identified weak similarity to class ii fusion proteins , revealing a segment within a cysteine - rich portion of  hap2 that could potentially correspond to the fusion loop .
we demonstrate by mutational analysis and fusion - blocking antibodies targeting this segment that it has elements that are essential for hap2 function .
finally , we show that the recombinant hap2 ectodomain is monomeric , but inserts into liposomes by concomitantly forming trimers , the x - ray structure of which revealed a class ii fusion protein fold in the typical trimeric post - fusion 
hap2 has 16 conserved cysteine residues with a signature distribution in the ectodomain ( figure  1b ) .
early alignments of  hap2 family members identified a characteristic 50 aa domain ( residues 352399 in chlamydomonas
hap2 ) with several conserved residues that was designated the hap2/gcs1 pfam domain ( pf10699 ) ( see http://pfam.xfam.org/family/pf10699 ) ( finn et  al . , 2016 ) . a previous mutagenesis analysis in chlamydomonas failed to identify functional properties in the pf10699 domain , as the mutant proteins tested either were not transported to the mating structure , or were nearly indistinguishable from wild - type in their ability to support fusion with plus gametes ( liu et  al . , 2015 ) .
database searches for additional conserved regions using the hhpred protein homology detection server ( sding et  al . , 2005 ) indicated that a cysteine - rich region in the n - terminal half of the ectodomain exhibited weak similarity to class ii fusion proteins .
in particular , hhpred identified a polypeptide segment in c.  reinhardtii hap2 ( 170204 , sqvwddtfgsskertranldcdfwsdpldiligrk ) that fell in the fusion loop region of the flavivirus envelope protein e in the resultant amino - acid - sequence alignment ( figure  s1 ) .
analysis of hap2 orthologs showed that the sequence in this region is highly variable , with a number of deletions and insertions and is framed at each side by relatively conserved segments : amino acids ( aa ) 159167 upstream ( including conserved cysteines 57 ) and aa 208219 downstream ( including conserved cysteine 9 ) ( figures 1b and 1c ) . only amino acids r185 and
c190 ( in bold  in  the sequence above ) within the identified segment are  conserved , suggesting that they may play a role in hap2 function . to investigate the functional importance of this segment we transformed chlamydomonas wild - type ( wt ) or mutant hap2 transgenes carrying an influenza virus hemagglutinin tag ( ha ) into a fusion - defective , hap2 mutant strain ( liu et  al . , 2008 ) and assessed hap2-ha expression and trafficking to the mating structure as well as fusion of the transformed hap2  minus gametes with wt plus gametes .
hap2-ha was detected in hap2  minus gametes transformed with wt hap2-ha as the expected doublet in sds - page / immunoblotting ( figure  1d ) , the upper form of which was present on the cell surface as assessed by its sensitivity to protease treatment of live gametes ( liu et  al . , 2008 ) .
all mutant proteins were expressed at levels similar to wild - type hap2-ha ( figure  1d ) , trafficked to the cell surface as assessed by their sensitivity to trypsin treatment of live gametes ( examples shown in figure  1e ) , and localized at the mating structure ( example shown in figure  1f ) .
thus , any defects in gamete fusion could be ascribed directly to the functional properties of  the mutant hap2 proteins .
hap2 with a deletion of residues 184tra186 ( hap2-tra - ha ) , which includes the conserved r185 was non - functional and failed to rescue fusion in the hap2 mutant when mixed with wild - type plus gametes ( figure  1d ) .
a  mutant hap2 with a lysine sustituted for the conserved r185 ( the hap2-r185k - ha mutant ) was fully functional , whereas the  hap2-r185a - ha or hap2-r185q - ha mutants failed to rescue fusion ( figure  1d ) .
a reverse - order hap2-ra185 - 86 mutant ( hap2-r185a - a186r - ha ) also was non - functional , indicating that a positively charged residue at position 185 is essential for the fusion activity .
finally , hap2 minus gametes expressing hap2-f192a - w193a - ha were impaired in fusion , although fusion was not abolished by these mutations , indicating that these nearby aromatic residues also play a role in hap2 fusion function ( figure  1d ) .
thus , the segment hap2170 - 204 , bounded by a pair of conserved cysteines , contains residues that are dispensable for protein expression , folding and localization , but are essential for the membrane fusion activity . in an independent approach to examine the function of the hhpred - identified region , we generated a rabbit antibody against a synthetic peptide , hap2168 - 190 , spanning the functionally important r185 residue .
the affinity - purified antibody ( -hap2168 - 190 ) immunoprecipitated epitope - tagged hap2-ha from lysates of hap2-ha minus gametes ( figure  2a ) , confirming its reactivity with hap2 . to test whether -hap2168 - 190 interfered with gamete fusion , we incubated minus gametes with undiluted antibody , mixed them with plus gametes , and determined the percentage of gametes that had fused to form zygotes
pre - incubation of minus gametes with -hap2168 - 190 had no effect on motility or adhesion but inhibited gamete fusion  by over 75% , whereas pre - incubation with a control igg had no effect on fusion ( figures 2b and 2c ) .
antibody dilution resulted in a loss of fusion - blocking activity , suggesting a low concentration of hap2-specific antibodies in the polyclonal mixture , probably due to a low immunogenicity of the synthetic peptide .
pre - incubation of plus gametes with the antibody did not affect their ability to fuse ; and the fusion - blocking activity of -hap2168 - 190 was neutralized by pre - incubation with the hap2168 - 190 peptide , but not with a control peptide ( figure  2c ) , further documenting the specificity of the antibody .
finally , immunolocalization experiments using an anti - ha antibody showed that hap2-ha on -hap2168 - 190-treated minus gametes remained at the mating structure ( figure  2d ) , indicating that -hap2168 - 190 did not alter hap2 localization , but directly interfered with its function .
these functional studies with chlamydomonas mutant gametes and the anti - peptide antibody indicated that in its native conformation on live gametes , the 168190 segment of hap2 is accessible at the protein surface and its integrity and availability are essential for fusion of gametes .
we used a drosophila expression system to produce a soluble form of c.  reinhardtii hap2 aa 23592 ( comprising almost the entire ectodomain , figure  1b ) and purified it to homogeneity ( see star methods ; figure  s2 ) .
analysis by size - exclusion chromatography ( sec ) and multi - angle static laser light - scattering ( malls ; figure  s2 ) showed that the protein behaved as a monomer in solution ( fraction labeled hap2e ) , but had a tendency to oligomerize with time ( especially under high ionic strength conditions ) to elute at a volume corresponding roughly to hexamers ( hap2eh fraction in figure  s2 ) .
the purified protein from the hap2e monomeric fraction efficiently neutralized the fusion - inhibition potential of the -hap2168 - 190 antibody ( figure  2c ) , indicating that at least part of the 168190 segment is exposed in hap2e and accessible to the antibody . to detect membrane insertion , we incubated the recombinant protein with liposomes of a standard lipid composition ( see star methods ) and monitored binding by co - flotation on a sucrose gradient ( figure  3e ) followed by immunoblot detection of lipid - inserted hap2e using a monoclonal antibody raised against hap2e ( mab k3 ; see figure  s2 ; star methods ) .
we observed efficient co - flotation of the monomeric hap2e fraction ( figure  3e ) , but not of the multimeric hap2eh fraction ( not shown ) .
electron microscopy analysis showed that hap2e decorated the liposome surface as projecting rods about 12  nm long ( figures 3b3d ) , which are similar to those formed by viral class ii fusion proteins in their trimeric , post - fusion form , such as the alphavirus e1 protein ( gibbons et  al . , 2003 ) .
the size and shape of  membrane - bound hap2e suggested that it had also oligomerized upon membrane insertion . of note ,
3-fold symmetry was  apparent in some top views of unbound proteins present in the background ( figure  3d , arrowheads ) .
we confirmed that hap2e had indeed trimerized upon membrane insertion by detergent - solubilizing it from the liposomes and analyzing it by native page ( figure  3f ) and by sec ( figure  s3 ) .
these results indicated that hap2e behaves similarly to the alphaviruses and flaviviruses class ii proteins , with membrane insertion concomitant with trimerization of a monomeric pre - fusion form ( klimjack et  al .
2002 ) , except that hap2e did not require an acidic environment for lipid binding and trimerization .
this difference is in line with the ability of hap2 to induce gamete fusion in the extracellular environment , whereas alpha- and flaviviruses require the acidic environment of an endosome for fusion . because the co - flotation experiments demanded relatively high amounts of purified recombinant protein for detection , it was impractical to assess flotation inhibition by the undiluted polyclonal -hap2168 - 190 antibody .
but we tested the effect of the mutations described in figure  1 by recombinantly expressing the mutant hap2 ectodomains .
these experiments showed that the mutations that impaired gamete fusion also affected the co - flotation capacity of the mutant hap2e ( figure  3 g ) .
the r185k hap2e mutant co - floated with liposomes as efficiently as wild - type , whereas those in which the charge at position 185 was removed  the r185a and r185q mutants  co - floated poorly ( figure  3 g ) .
co - flotation with the liposomes was also essentially abrogated with the hap2e-tra and hap2e - f192a - w193a mutants .
the combination of the in  vivo ( figures 1 and 2 ) and in  vitro ( figure  3 ) analyses indicates that hap2 has the capacity to directly interact with membranes and that altering the conserved residues of the hhpred - identified segment affects this interaction .
details of the crystallization and structure determination are described in the star methods , and the crystallographic statistics are listed in table s1 .
we determined the x - ray structure by the single isomorphous replacement with anomalous scattering ( siras ) method using a ptcl4 derivative .
the experimental electron density map allowed the tracing of 442 amino acids out of 569 in the hap2e expression construct ( aa 23592 ; figure  1b ) from amino acids 24 to 581 with internal breaks at several disordered loops ( listed in the star methods ) .
the atomic model of c.  reinhardtii hap2 revealed a trimer with unambiguous structural homology to class ii fusion proteins , featuring the three characteristic  sheet - rich domains , termed i , ii , and iii , arranged in a 
hairpin  conformation typical of class ii fusion proteins in their post - fusion form ( bressanelli et  al . , 2004 ,
modis et  al . , 2004 , willensky et  al . , 2016 )
the overall shape of the hap2e trimer is totally compatible with the projections from the liposomes observed in the electron micrographs , with the predicted membrane interacting region at the tapered end of the rods ( figures 3c and 3d ) .
the monomeric hap2e ( figure  s2 ) used to grow the crystals obviously underwent the same conformational change to form trimers observed during insertion into liposomes ( figure  3 ) .
a  similar oligomeric rearrangement during crystallization was reported previously for the dengue virus e protein under acidic ph conditions ( klein et  al . , 2013 , nayak et  al . , 2009 ) .
in the case of hap2 , acidification is not required ; the  trigger for the rearrangement to the post - fusion form is not known .
the comparison with class ii fusion protein trimers of known structure defines the membrane - facing side ( top in figure  4 ) and the membrane distal end ( bottom side ) of the hap2 trimer , in agreement with the shape observed in the electron micrographs ( figures 3b3d ) .
the dali server ( holm and park , 2000 ) yielded z scores ranging from 9 to 16 for up to 343 c atoms of class ii fusion proteins of viral and cellular origin ( table 1 ) , in the same range as those obtained in previous comparisons among known class ii fusion proteins ( prez - vargas et  al . , 2014 ) .
domain i in hap2 is a  sandwich of about 200 aa with ten  strands in the two apposed  sheets : the a0b0i0h0g0 sheet is buried and the j0c0d0e0f0 sheet is exposed in the trimer , and we refer to them as inner and outer sheets ( figures 5a and 5d ) .
a specific feature of the hap2 domain i is that the a0b0 -hairpin is long and projects out of the inner sheet to augment the outer sheet of the adjacent subunit , where strand a0 runs parallel to j0 ( figure  s5 ) .
these additional inter - subunit main chain -interactions are likely to confer extra stability to the post - fusion trimer . at the membrane - distal ,
bottom face of the trimer , the outer  sheet projects very long loops , which are disordered in the crystal ( dashed tubes in figures 4 , 5 , s4 , and s5 ) .
the c0d0 loop projection displays six additional cysteine residues ( presumably forming three disulfide bonds ) in an insertion that is present essentially only in algal hap2 ( liu et  al . , 2008 )
( figure  5d ) ; the  e0f0 loop projection is also an insertion with four potential n - linked glycosylation sites .
domain iii is made of about 130 aa and has an immunoglobulin - like fold , with seven  strands in two sheets ( labeled abe and gfcc ; figures 5a , 5d , and s4 ) . although the  sheets are longer than in other class ii fusion proteins ( figure  4 ) , giving domain iii a more elongated bean shape , it is in a similar location  at the side of the trimer ( figure  4 ) as in other class ii fusion proteins of known structure .
core ,  which is composed of domains i and ii arranged parallel to each other and interacting along their length about the 3-fold molecular axis .
the latter central trimer interaction is postulated to exist during formation of an  extended trimeric intermediate 
2010 ) , in which the fusion loops are inserted in the target membrane ( plus gamete here ) , while the c - terminal tm segment is anchored in the minus gamete mating structure , at the opposite end .
the final collapse into the post - fusion hairpin conformation of each protomer in the trimer brings domain iii to the sides of this core , projecting the downstream stem and tm regions toward the fusion loop ( liao and kielian , 2005 ) , in a fusogenic rearrangement of the protein analogous to an umbrella folding inside out . in this final , post - fusion location
, domain iii buries an area of about 2,100   of its surface , divided roughly equally in contacts with the same and with the adjacent subunit ( intra- and inter - subunit contacts ) .
the observed contacts therefore can only form after the assembly of the central trimer core , in line with the proposed clamping role of domain iii , and resulting in irreversible trimerisation , as proposed for other class ii fusion proteins ( liao and kielian , 2005 , prez - vargas et  al . , 2014 ) .
domain ii is the largest domain ( roughly 250 aa in total ) and is made by two distinct segments emanating from domain i : the d0e0 and h0i0 strand connections of the outer and inner sheets of domain i , respectively ( figure  s4 ) .
as in all class ii proteins , the domain i proximal region of domain ii has a central  sheet ( aefg ; figures 5 and s4 ) flanked by additional short helices .
the distal  tip  of domain ii contains  sheet bdc , with the   strands running parallel to the molecular 3-fold axis at the distal end of the d0e0 segment .
the connection between  strands c and d at the tip of domain ii ( the cd loop ) was shown to be the fusion loop in the viral proteins ( reviewed in kielian and rey ) .
the bdc sheet normally packs against the ij -hairpin ( the distal end of the h0i0 segment ) , which in hap2 maps to the conserved hap2/gcs1 pfam domain pf10699 ) ( figure  5a ) .
although in hap2  strands i and j are absent , we still refer to this region as the  ij loop  ( figures 5 and s4 ) .
the cd loop in hap2 is 40 aa long and has an intervening short -helix in the middle ( 0 ) , in contrast to the standard class ii fusion proteins from the arthropod - borne viruses such as flaviviruses and alphaviruses ( 1015 residues long ) . in this respect
, hap2 resembles the rubella virus class ii fusion protein e1 , which has 50 aa in between stands c and d , with a couple of short intervening  helices as well as an additional  strand which results in two separate fusion loops ( dubois et  al . , 2013 ) .
the presence of the 0 helix in hap2 also results in two loops ( loops 1 and 2 in figures 5b and 5c ) , which project outward .
although disordered in the crystal , these two loops are in position to project non - polar residues into the target membrane .
the hhpred alignment was indeed quite close and pointed correctly to the cd loop ( figure  s1 ) .
the hap2168 - 190 peptide used for immunization spans loop 1 all the way to the end of helix 0 , and the fact that the resulting polyclonal antibody blocks fusion is in line with this region being exposed at this end of the molecule .
the hap2 crystal structure is therefore compatible with the mutagenesis data in this region , and with the effect of the -hap2168 - 190 antibody .
moreover , a peptide derived from the corresponding region of  tetrahymena thermophila hap2 was found to display properties typical of a fusion loop ( see related paper in current biology , pinello et  al . , 2017 ) , in line with our findings with c.  reinhardtii .
the structure shows that the functionally important residue r185 is at the n terminus of the 0 helix and its side chain points away from the two exposed loops and toward the core of the hap2 trimer .
r185 makes a salt bridge and bidentate hydrogen bonds with the side chain of the strictly conserved e126 in  strand b  ( figures 5b and 5c ) .
furthermore , the r185 side chain is at the core of a network of interactions stabilizing the main - chain conformation of the ij loop .
this network involves main chain atoms of f376 , g382 and r385 , together with the strictly conserved q379 side chain , which are part of the pf10699 signature segment that allowed the identification of hap2 in widely disparate organisms .
the structure now shows that the conserved pattern of disulfide bonds of this signature element is required for the ij loop to adopt a convoluted fold acting as a framework  underpinning the cd loop by interaction with the conserved r185 so that the two fusion loops project out at the membrane - interacting region .
the latter region , in contrast , is variable and has multiple deletions and insertions in the various orthologs , some insertions being quite long ( figure  1c ) .
it is possible that the differences in membrane - interacting regions of hap2s across the broad spectrum of eukaryotic organisms reflect evolutionary adaptations required for fusion with different target gametes .
we note that the fusion loop in viral class ii proteins is in general the most conserved segment of the protein within orthologs from a given virus genus , most likely because the same residues are also required for inter - subunit interactions in their pre - fusion form on infectious particles . but this comparison is not necessarily informative , since the analyzed hap2 proteins span eukaryotic taxa that are much more distantly related than are viruses within a given genus .
the pre - fusion conformation of hap2 remains unknown , however , and it is possible that the non - polar residues of the two fusion loops are maintained unexposed until the time of fusion
. the conserved interaction of the r185 side chain with that of the e126 residue in the tip domain may allow exposure of the two fusion loops only after a conformational change . in this context
, the fusion - inhibiting -hap2168 - 190 antibody would bind only after a conformational change that exposes the fusion loop , but this remains to be explored . the identical topological arrangement of secondary , tertiary , and quaternary structure elements of hap2 with the viral class ii proteins
indeed , the probability of convergence to the observed complex fold from independent origins , to result in two proteins displaying exactly the same topological arrangement throughout the entire ectodomain ( as shown in figure  s4 ) is extremely low and can be considered negligible .
nature is parsimonious , and once a protein required for a complex function such as membrane fusion becomes available , the corresponding gene is used over and over again , most likely transferred via horizontal gene exchanges .
this concept is supported by the observation that only three structural classes of viral fusion proteins have been observed so far , in spite of the enormous variety of known viruses .
as one example , the membrane fusion proteins of the herpesviruses , rhabdoviruses and baculoviruses , which are otherwise totally unrelated viruses , were shown to be homologous by structural studies ( heldwein et  al . , 2006 , kadlec et  al . , 2008 , roche et  al . , 2006 ) .
these fusion proteins most certainly derived from a distant common ancestor ( class iii proteins ) ( backovic and jardetzky , 2011 ) , whose genes must have been acquired via horizontal exchanges . within eukaryotic organisms , only a few other cell - cell fusion proteins have been positively identified .
although the myoblast myomaker , a seven - pass transmembrane protein that governs fusion of myoblasts to form myotubes ( millay et  al . , 2013 , millay et  al . , 2016 ) , has no obvious relation to viruses , the proteins involved in the two other cell - cell fusion events that have been characterized at the molecular level are virus related .
cytotrophoblast fusion in mammals during placenta formation ( blaise et  al . , 2003 ,
holm and park , 2000 ) and epidermal cell - cell fusion in nematodes to form syncytia ( mohler et  al . ,
2002 ) both use fusion proteins also found in viruses . in the first case ,
the class i fusion protein involved is clearly derived from an endogenous retroviral element ( denner , 2016 ) .
it is also possible that a similar process involving retrotranscription may have taken place in the case of the class ii caenorhabditis elegans fusion protein eff-1 ( prez - vargas et  al . , 2014 ) , as retroviruses of nematodes have been found to have an envelope protein related to that of the phleboviruses ( frame et  al . , 2001 ,
, 2000 ) , which have class ii fusion proteins ( dessau and modis , 2013 ) .
these observations suggest that retro - transcription , followed by integration into the genome , may have been an important pathway for gene exchanges between viruses and eukaryotic cells .
hap2 is present in organisms responsible for several of the globe s most devastating human diseases , including plasmodium , trypanosoma , and toxoplasma .
many arthropods that are vectors of human diseases or are agricultural pests , such as insects and ticks , also possess hap2 . a strategy that used recombinant plasmodium hap2 fragments to induce transmission - blocking immunity in mice
has been reported previously ( blagborough and sinden , 2009 , miura et  al . , 2013 ) , but the expression systems were not sufficient for viable clinical developments .
our results suggest that the use of a peptide spanning the hap2 fusion loops as immunogen might be sufficient to induce transmission - blocking immunity , similar to the antibodies we obtained here against chlamydomonas hap2 . in conclusion
, our data now open the way to a full mechanistic characterization of gamete fusion induced by hap2 and raise new questions , including the identification of the trigger for the hap2 fusogenic conformational change , the structure of the pre - fusion form(s ) of hap2 , and its organization on the mating structure of the minus gamete membrane .
evolution through hundreds of millions of years may have led the different taxa to develop alternative solutions , and the metastable pre - fusion form of hap2 may be organized differently in the multiple organisms in which it is present , as shown for viruses of different families . in contrast , the post - fusion conformation described here appears as a universal feature of class ii fusion proteins .
reagent or resourcesourceidentifierantibodiespolyclonal rabbit -hap2168 - 190 peptide abthis papern / amonoclonal mouse -hap2e ab k3this papern / achemicals , peptides , and recombinant proteinshap2168 - 190 ( sssqvwddtfgsskertranldc)yenzym antibodiesn / ahap2control ( ctqpprppwpprpppappps)yenzym antibodiesn / acommon lab reagentsn / an / adeposited datac . 
melanogaster : cell line s2 : s2thermo - fishercat # r690 - 07experimental models : organisms / strainschlamydomonas reinhardtii : strain 21 gr : mt+chlamydomonas culture collectioncc-1690chlamydomonas reinhardtii : strain 40d4 : hap2 mating type minus mutantchlamydomonas culture collectioncc-4552chlamydomonas reinhardtii : strain hap2-ha : hap2-ha plasmid - rescued 40d4 strainchlamydomonas culture collectioncc-5295recombinant dnapmt / bip / twinstrepkrey et  al .
- 592twinstrepthis papern / asoftware and algorithmsxdskabsch , 1988http://xds.mpimf - heidelberg.mpg.de / pointlessevans , 2006http://www.ccp4.ac.uk / ccp4collaborative computational project , 1994http://www.ccp4.ac.uk / autosharpvonrhein et  al .
, 2010https://www.globalphasing.commolprobitychen et  al . , 2010http://molprobity.biochem.duke.edu / hhpredsding et  al . , 2005https://toolkit.tuebingen.mpg.de / hhpreddaliholm and park , 2000http://ekhidna.biocenter.helsinki.fi / dali_server / start requests for resources and reagents should be directed to flix a. rey ( felix.rey@pasteur.fr )
. wild - type chlamydomonas strains 21 gr ( mating type plus ; mt+ ; cc-1690 ) , 40d4 ( hap2 mating type minus mutant ( liu et  al . , 2015 ) ) and hap2-ha ( hap2-ha plasmid - rescued hap2 strain ( liu et  al . , 2015 ) ) were used and are available from the chlamydomonas culture collection
. vegetative growth of cells and induction of gametogenesis in gamete medium ( m - n ) were as described before ( liu , et  al .
gametes were activated with dibutyryl - camp ( db - camp ) by incubation with 15  mm db - camp and 0.15  mm papaverine for 0.5  hr in n - free medium ( ning et  al . , 2013 ) .
gamete fusion was assessed by determining the number of cells that had formed zygotes after being mixed with wild - type plus gametes for 30  min and was expressed as percent fusion using the following equation : ( 2   number of zygotes)/[(2   number of zygotes)+(number of unfused gametes ) ]  100 ( liu et  al . , 2015 ) . a polyclonal antibody against hap2 peptide sssqvwddtfgsskertranldc ( aa 168 - 190 ) was made in rabbits by yenzym antibodies ( san francisco , ca ; -hap2168 - 190 ) , under oversight by their institutional review board .
the antibody was purified on a peptide - conjugated affinity column prepared by the company . to assay for antibody inhibition of gamete fusion , gametes that had been activated with dibutyryl - camp for 30  min ( misamore et  al . , 2003 )
were washed once with gamete medium and the activated gametes ( 2   10 cell / ml ) were incubated with 110 ug / ml peptide antibody ( pre - dialyzed in gamete medium ) for 2  hr followed by mixing with gametes of the opposite mating type gametes for the times indicated in the figure legends .
the number of zygotes ( detected as cells with 4 rather than 2 cilia ) was determined by phase - contrast microscopy .
at least 100 cells were counted each time with at least two counts per sample in at least two separate experiments .
a rabbit igg ( sigma ) was used as a control antibody . to test the capacity of peptides or hap2e to neutralize the fusion - inhibiting properties of -hap2168 - 190 ,
the antibody ( 220 ug / ml ) that had been dialyzed in gamete medium was incubated overnight with control peptide ctqpprppwpprpppappps ( 200 ug / ml ) ( this peptide is encoded by cre03 g176961 whose transcripts are specific to minus gametes and upregulated during gamete activations ( ning et  al . , 2013 ) , hap2168 - 190 peptide ( 200 ug / ml ) , or hap2e protein ( 200 ug / ml ) overnight and then added to activated gametes as above to determine percent gamete fusion .
immunofluorescent staining of gametes was performed as described previously ( belzile et  al . , 2013 ) .
the gametes were fixed in ice cold methanol ; the primary antibody was rat anti - ha ( roche ) and the secondary was alexa fluor 488 goat anti - rat ( invitrogen ) .
images were captured in dic or fitc channels as described previously ( liu et  al . , 2015 ) .
for hap2-ha localization after pre - incubation in -hap2168 - 190 ( see below ) , db - camp - activated gametes were incubated with the antibody for 2  hr , washed three times with gamete medium , and stained with ha antibody as above .
for immunoprecipitation , gametes were disrupted by incubation at 4c for 30  min in ripa buffer ( 20  mm tris , 150  mm naci , 1% np-40 , 0.5% deoxycholate , 0.1% sds ) containing a proteinase inhibitor cocktail ( roche ) .
the samples were centrifuged at 12000  rpm for 30  min , the supernatants were subjected to immunoprecipitation with -hap2168 - 190 and protein a agarose beads using methods described previously ( liu et  al . , 2010 ) and the immunoprecipitated proteins were subjected to sds - page and immunoblotting ( liu et  al . , 2015 ) . for trypsin treatment 5x10 live gametes
/ ml were incubated in 0.05% freshly prepared trypsin for 20  min at room temperature , diluted 10-fold with n - free medium , centrifuged , and resuspended in fresh n - free medium containing 0.01% chicken egg white trypsin inhibitor . for immunoblotting ,
the remaining cells were washed twice more with n - free media containing 0.01% chicken egg white trypsin inhibitor before analysis by sds - page and immunoblotting ( misamore et  al . , 2003 ) .
tra184 - 6 , r185a , r185k , r185q , ra185 - 6ar and fw192 - 3aa mutant forms of chlamydomonas hap2 ( gi:288563868 ) were performed using standard pcr methods .
the pcr fragments were inserted into the hap2-ha plasmid at bglii / nrui sites using in - fusion dry - down pcr cloning kit ( clontech ) .
plasmids were transformed into the 40d4 hap2 mutant using electroporation and selected on paromomycin plates ( liu et  al . , 2015 ) .
colonies were selected based on pcr identification of the transgene and confirmation of hap2-ha expression by immunoblotting ( liu et  al . , 2008 ) .
codon - optimized synthetic cdna corresponding to a soluble c - terminally truncated version of the hap2 ectodomain ( hap2e ) comprising residues 23 - 592 from chlamydomonas reinhardtii was cloned into a modified drosophila s2 expression vector described previously and transfection was performed as reported earlier ( krey et  al . , 2010 ) .
for large - scale production , cells were induced with 4  m cdcl2 at a density of approximately 7x10 cells / ml for 8  days , pelleted , and the soluble ectodomain was purified by affinity chromatography from the supernatant using a streptactin superflow column followed by size exclusion chromatography using a superdex200 column in 10 mm bicine ph9.3 .
purified hap2e ectodomain was subjected to sec using a superdex 200 column ( ge healthcare ) equilibrated with the indicated buffers .
online malls detection was performed with a dawn - heleos ii  detector ( wyatt technology , santa barbara , ca , usa ) using a laser emitting at 690  nm .
online differential refractive  index measurement was performed with an optilab t - rex detector ( wyatt technology ) .
data were analyzed , and weight - averaged  molecular masses ( mw )  and mass distributions ( polydispersity ) for each sample were calculated using the astra software ( wyatt technology ) .
balb / c mice were immunized subcutaneously with 10  g of recombinant hap2e in complete freund s adjuvant and boosted five times with the same antigen dose in incomplete freund s adjuvant .
mice splenocytes were fused to p3u1 myeloma cells and growing hybridomas were selected in an elisa test on plates coated with 1  g / ml hap2e .
monoclonal antibody k3 was purified using protein g hitrap columns ( ge healthcare ) according to the manufacturer s instructions followed by sec in pbs using a sdx200 column .
dope ( 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine ) , dopc ( 1,2-dioleoyl- sn - glycero-3-phosphocholine ) , cholesterol and sphingomyelin were purchased from avanti polar lipids .
liposomes were freshly prepared by the freeze  thaw and extrusion method using molar ratios of 1/1/3/1 of dope / dopc / cholesterol / sphingomyelin .
0.7  m purified hap2e were mixed with 8  mm liposomes and incubated 1h at 25c in 100  l pbs .
samples were then adjusted to a final concentration of 20% sucrose , overlayed with a 5%60% sucrose gradient ( in pbs ) and centrifuged overnight at 4c at 152.000 x g. fractions from the top , middle and bottom of the gradient were analyzed by immunoblotting using specific anti - hap2 monoclonal antibodies and the bands quantified using the genetools syngene software .
the percentage of hap2e in either fraction was calculated as the ratio between hap2e in individual fractions and total hap2e ( sum of hap2e in top and bottom fractions ) .
purified hap2e ( c.  reinhardtii ) mixed with liposomes was spotted on glow discharged carbon grids ( cf300 , ems , usa ) , negatively stained with 2% phosphotungstic acid ( pta ) ph 7.4 , analyzed with a tecnai g2 bio - twin electron microscope ( fei , usa ) and imaged with an eagle camera ( fei , usa ) . for cryo - electron microscopy liposomes
alone or liposomes mixed with purified hap2e were applied on a glow discharged lacey carbon grid ( agar scientific , uk ) .
samples were plunge - frozen in liquid ethane using an automated system ( leica emgp , austria ) and visualized on a tecnai f20 electron microscope operating at a voltage of 200 kv .
image frames were recorded in low - dose mode on a falcon ii direct electron detector ( fei , usa ) .
purified recombinant hap2e in pbs and hap2e from the top fraction of the sucrose gradient solubilized with 4% chaps were independently purified by size - exclusion chromatography using a superdex 200 increase column .
elution fractions corresponding to the  respective proteins were further analyzed on a 4%16% native gradient gel using the nativepage novex bis - tris gel system ( invitrogen ) followed by silver staining .
crystals of hap2e were obtained using in  situ proteolysis as described before ( dong et  al . , 2007 ) .
briefly , subtilisin dissolved in 10 mm tris ph8 , 30 mm nacl at 10mg / ml was added to protein solution ( 12 - 14  mg / ml in 10  mm bicine , ph 9.3 ) on ice immediately prior to crystallization trials in a 1/100w : w ratio
. crystals of hap2e were grown at 293k using the hanging - drop vapor - diffusion method in drops containing 1  l protein / protease solution mixed with 1  l reservoir solution containing 100  mm hepes ph7.5 , 2% 2-propanol , 100  mm sodium acetate , and 12%14%w / v peg 8000 .
diffraction quality rod - like crystals appeared after 1  week and were flash - frozen in mother liquor containing 30% ( v / v ) mpd .
data collection was carried out at the swiss light source ( px i ) , the esrf ( id30a-3 ) , and the synchrotron soleil ( proxima1 ) .
data were processed , scaled and reduced with xds ( kabsch , 1988 ) , pointless ( evans , 2006 ) and programs from the ccp4 suite ( collaborative computational project , 1994 ) . a single - wavelength anomalous dispersion ( sad ) dataset
was collected from a single crystal of hap2e from c.  reinhardtii soaked for 6  hr in 2  mm k2ptcl4 solution in cryo buffer .
data were collected at the liii edge of platinum ( 1.072   ) on a single crystal using low - dose ( 0.5 mgy per 360 ) , high - redundancy ( 5   360 ) fine -sliced collection strategy using five crystal orientations by means of a high - precision multi - axis prigo goniometer ( weinert et  al . , 2014 ) .
an initial set of experimental phases was obtained by the single isomorphous replacement method using autosharp ( vonrhein et  al . , 2007 ) with the platinum derivative and a highly isomorphous native dataset .
starting phases were improved by consecutive cycles of manual building and combination with phases derived from molecular replacement using the partial model as search model in phaser ( mr - sad ) ( mccoy et  al . , 2007 ) . after building an initial poly - alanine model accounting for 50% of the c atoms these phases were further refined using  the anomalous signal of a highly redundant sulfur - sad dataset collected at a wavelength of 2.06641   on crystals of the native  protein following a similar collection strategy as mentioned above ( weinert et  al . , 2014 ) .
model building was performed using  coot ( emsley et  al . , 2010 ) , and refinement was done using autobuster ( bricogne et  al . , 2010 ) with repeated validation using  molprobity ( chen et  al . , 2010 ) .
the final model includes amino acids 24 to 581 ( see linear diagram in figure  1b ) , with internal breaks at loops 69 - 97 , 152 - 156 , 167 - 182 , 194 - 205 , 238 - 283 and 330 - 345 , corresponding to disordered loops that are marked with  a  gray background in the c.  rheinhardtii sequence in figure  5d ( top sequence ) and as dashed tubes in the ribbon diagrams ( figures 4 and 5a5c ) .
clear electron density was observed for one n - linked and one o - linked glycan chain ( attached to n497 and t577 in domain iii ) .
data are presented as mean  sd unless otherwise indicated in figure legends and experimental repeats are indicated in figure legends .
the accession number for the atomic coordinates and structure factors reported in this paper is pdb : 5mf1 .
designed the mutant hap2 proteins and selected the peptide used to produce the fusion - blocking antibody . w.j.s . , y.l . , and w.l .
performed the gamete fusion assays with hap2 mutants and antibodies and interpreted them with w.j.s .
collected data and determined the hap2 x - ray structure and g.b . helped devise a collection strategy for derivative x - ray data .
 summarysexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell . 
 the sperm - restricted single - pass transmembrane protein hap2-gcs1 has been postulated to function in membrane merger . 
 its presence in the major eukaryotic taxa  animals , plants , and protists ( including important human pathogens like plasmodium)suggests that many eukaryotic organisms share a common gamete fusion mechanism . here , 
 we report combined bioinformatic , biochemical , mutational , and x - ray crystallographic studies on the unicellular alga chlamydomonas reinhardtii hap2 that reveal homology to class ii viral  membrane fusion proteins . 
 we further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks  gamete  fusion . 
 these results demonstrate that hap2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion , indicating a way to block plasmodium transmission and highlighting the impact of virus - cell genetic exchanges on the evolution of eukaryotic life . 
Introduction
Results and Discussion
STARMethods
Author Contributions