Inhibition mechanism of the fungal β−1,3-glucan synthases by triterpenoid antifungal drugs

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: minor_revision

Abstract
β−1,3-glucansynthaseisthemoleculartargetfortriterpenoidandechinocandinantifungaldrugsinclinical.Itcatalyzestheformationofβ−1,3-glucan,whichistheprimarycomponentofthefungalcellwall.However,theinhibitionmechanismofβ−1,3-glucansynthasebytriterpenoiddrugsremainsunclear.Inthisstudy,wereportcryo-electronmicroscopy(cryo-EM)structuresofSaccharomycescerevisiaeβ−1,3-glucansynthaseFks1andFks2intheapostate,thetriterpenoiddrugenfumafungin-boundstate,andanopenstate.Structuralanalysisalongwithmutagenesisrevealstheenfumafunginbindingsite,andthemechanismoftheclinicaldrug-resistantmutationsoftheβ−1,3-glucansynthases.Remarkably,theenfumafunginattachesonasingletransmembranehelixTM5oftheβ−1,3-glucansynthases,reorganizesitsnearbylipidenvironment,andstabilizestheenzymeinaspecificbasalstatewithintactactivesite.Moreover,weelucidatethatboththebasalstateandtheopenstateareessentialforFKS’sglycosyltransferaseactivity.OurresearchalsoshowsthatFks2ishighlyconservedwithFks1intermsofstructure,activity,anddruginhibition.Thesefindingsprovidedeepinsightsintothefungalcellwallsynthesis,andwillfacilitatethedevelopmentofantifungaldrugstargetingβ−1,3-glucansynthase.

Introduction
FungiareamongthemostdiversegroupsoforganismsonEarth,withan estimated over 1.5 million species1. Among them, approximately 8000speciescauseplantdiseases,andmorethan300speciescause humandiseases2. Every year, diseases related to fungal infections affectapproximately300millionpeopleworldwide,resultinginabout 1.6milliondeaths3,4. Duetothelimitedavailabilityofclinicalantifungal drugs and the continuous emergence of resistant pathogens, the expansion of the fungal infections necessitates urgent attention to developingdistincttherapeuticstrategies4–6.

β-1,3-glucansynthaseisapivotalintegralmembrane enzyme in fungal cells, catalyzing the synthesis of β-1,3-glucan, a fundamentalcomponentofthefungalcellwallthatcontributesto cell integrity and viability7,8. β-1,3-glucan synthase belongs to the glycosyltransferase48(GT48)family(http://www.cazy.org).Previous studieshavealsoshownthatRho1isessentialforactivityofthe1,3-β- glucansynthasebothinvivoandinvitro9–14. Usinguridinedi- phosphate-activated glucose (UDP-Glc) as the sugar donor, β-1,3- glucansynthasefacilitatesthecreationofβ(1→3)glycosidiclinkages in β-1,3-glucan and transports the glucan across the membrane (Fig.1a).Fungalβ-1,3-glucansynthaseisprimarilyencodedbyFKS1or FKS27.Expressionandfunctionofthetwogeneshavebeenshownto bedifferentialregulated7,15–19.InS.cerevisiae,FKS1isthepredominant isoformduringgrowth,whereasFks2isexpressedduringsporulation or under stress20. S. cerevisiae Fks1 and Fks2 share ~88% sequence identity. In S. cerevisiae, Candida albicans and many other patho- genicfungi,simultaneousknockoutofFKS1andFKS2islethal,while knockoutofFKS1aloneisalive8,21,22.MutationsinbothFks1andFks2 havebeenreportedtobeassociatedwithdrugresistance10–14.How- ever, whether Fks2 adopts a similar mechanism to Fks1 is unknownyet.

Sofar,structuresofS.cerevisiaeFks1intheapostateorincom- plexwithRho1,andanechinocandin-resistantmutantFks1(S643P)in complex with glucan product have been determined14,23,24. Among them,thecryo-EMstructureofFks1incomplexwithRho1showedthat Rho1 is positioned within a pocket between the glycosyltransferase domainofFKS1(GTdomain)andthetransmembranehelixspanning TM7–15. Structural analysis suggested Rho1’s GTP/GDP cycling may promoteadynamictransitionbetweendifferentstatesofFKS1,indu- cingtheproteinasamolecularpumptopushthegrowingglucanchain into FKS1’s transmembrane channel. However,the active sites in all availableFks1structureswerenotfullyresolved,asmanyimportant elements(such as theconservedhorizontalinterface helicesIF1/2/3 thatwereoriginallycharacterizedincellulosesynthase25)arepartially or totally invisible due to flexibility compared to the AlphaFold2 predicted model (Supplementary Fig. 1a–h). This has significantly hindered our understanding of the mechanism of β-1,3-glucan synthase.

Currently,therearetwotypesofclinicalantifungaldrugstarget- ing β-1,3-glucan synthase, including triterpenes (ibrexafungerp) and echinocandins (caspofungin, micafungin, anidulafungin, and rezafungin)22,26–29.Ibrexafungerpisasemisyntheticanalogofthenat- uraltriterpeneenfumafungin29–31.ItwasalsoanFDA(U.S.Foodand DrugAdministration)approveddruginadistinctantifungalclassfor morethan20years29.Notably,triterpeneswerehighlyactiveformany Fks1 mutant based echinocandin resistant strains, indicating its dif- ferent inhibitory mode comparing to echinocandins32–34. Despite its importance,structuralinsightsintothebindingmodeandmechanism ofthetriterpenedrugsremainunclear.

Here, we determined five different cryo-electron microscopy (cryo-EM)structuresofendogenousFks1andFks2intheapostate,in complex with enfumafungin, and anopen state induced by echino- candin incubation. These structures illustrate the enfumafungin binding site and an intact active site of β-1,3-glucan synthase, and depicttheconformationalchangeinducedbyechinocandinincuba- tion. Our results provide deep insights into the pharmacological mechanismsofβ-1,3-glucansynthases.

Results
StructureandcharacterizationofendogenousFks1andFks2
EndogenousFks1andFks2werebothpurifiedfromyeastcellswith C-terminal triple FLAG epitope tags. Because S. cerevisiae pre- dominantlyexpressedFks1inregularcondition8,theendogenousFks2 was purified from a FKS1 knock out strain. The mild detergent N- dodecyl-β-D-maltoside (DDM) and cholesteryl hydrogen succinate (CHS)wereusedtoextractproteinfromyeastmembrane,andlauryl maltoseneopentylglycol(LMNG)andCHSwereusedintheCryo-EM analysis.Thetaggedproteinswerepurifiedusingananti-FLAG(M2) affinitycolumnandasize-exclusionchromatography(SEC)(Supple- mentaryFig.2a,b).FunctionalcharacterizationwithinvitroUDP-Glo glycosyltransferaseassayconfirmed thatendogenous Fks1 and Fks2 bothshowedglycosyltransferaseactivityinthepresenceofRho1and GTPγS(Fig.1b,SupplementaryFig.2a,b).ItindicatesFks2alsouses Rho1asitsregulatorysubunit,likeFks1.Toconfirmthisfinding,we incubatedpurifiedFks2andRho1withcrosslinkerBS3andseparated thecrosslinked complexusinga size exclusion column. The shifted positionofthegelfiltrationpeakindicatedthecomplexofFks2-Rho1 was successfully purified (Supplementary Fig. 3a). We further per- formed EM analysis on this crosslinked complex, and the 2D class averagesclearlyshowedadditionaldensitycorrespondingtoRho1on Fks2(SupplementaryFig.3b).ThedetectedbindingpositionofRho1is consistent with the published structure of Fks1–Rho1 complex14. Together,theseresultssupportthatRho1isaregulatorysubunitfor bothFks2andFks1.

We performed single-particle cryo-EM on purified samples and obtainedcryo-EM3DmapsofendogenousFks1andFks2inapostates at3.2-Åand3.6-Åresolution,respectively(Fig.1c,d,Supplementary Figs.4and5).AtomicmodelsofendogenousFks1andFks2(named Fks1-apoandFks2-apo)werebuiltintothemapsandrefinedtogood statistics(SupplementaryTable1).TheoverallstructuresofFks1-apo (UniProt code P38631, 1876 residues) and Fks2-apo (UniProt code P40989, 1895 residues) are highly similar, with a root-mean-square deviation(RMSD)ofapproximately1.5Åover1449residues(Fig.1e), whichalignswiththeir~88%sequenceidentity.InbothFks1-apoand Fks2-apo structures, FKS is composed of a transmembrane domain (TMD)andalargecytosolicdomain(Fig.1c–e).TheTMDcomprises15 transmembranehelices(TM)andfouramphipathichorizontalhelices (HH1A,1B,2A,2B),whichcanbegroupedintotwoparts(TMD1and TMD2) with a sizable cavity between them. The cytosolic domain consists of two subdomains including an N-terminal domain (NTD; Fks1: Asn145 to Thr444; Fks2: Asn164 to Ser463) and a glycosyl- transferase(GT)domaininsertedbetweenTM6andTM7(Fks1:Ile713 toGln1281; Fks2:Ile732toGln1300)(Fig.1f).Theactivesite locates betweenGTandTMD,andtheglucantransportchannelislikelyright below the active site and corresponds to the cavity between TMD1 andTMD2.

Furthermore,structuresofFks1-apoandFks2-apodisplayseveral features that were not resolved before. We identified an element, HH1B,thatformsextensivehydrophobicinteractionswithTM9,TM14, andHH2A(Fig.1g).TheseinteractionshelptoimprovetheTMD2tobe morerigid.HH1BisconnectedwithHH1Abyashortloop(HH1-loop) (Fks1:Arg1512toSer1521;Fks2:Arg1531toSer1540),whichispartially flexibleinFks1butorderedinFks2.TheHH1-loopinFks2isstabilized byashorthelixrightbeforeHH2A.Inaddition,theexoplasmicloop connectingTM1andTM2wasmodeledinFks2,anditinteractsdirectly with the exoplasmic loop connecting TM7 and TM8, resulting in a moreorderedexoplasmicstructure(Fig.1h).

EnfumafunginbindingsiteandresistantmutationsofFKS
Enfumafungin(EFU)isatriterpeneglycosidethatwasproducedfrom thefungusHormonemasp30,31.Itfeaturesfoursix-memberedringswith four additional specific groups: glucose unit, acetate group, hemi- acetalmoiety,andcarboxylicacidgroup(Fig.2a)35.Previousstudies showedEFUdisplayedhighantifungalactivitywith<1μg/mlminimal inhibitoryconcentrations(MIC)againstvariousfungalpathogens,and its development has led to the semi-synthetic clinical drug, ibrexafungerp22,29.WedeterminedtheMICforEFUinS.cerevisiaetobe ~0.07μg/ml(Fig.2b).TounderstandhowEFUinteractsandinhibits FKS,wedeterminedthecryo-EMstructuresofFks1incomplexwith EFU(Fks1–EFU)andFks2incomplexwithEFU(Fks2–EFU)atareso- lution of 3.0-Å and 3.3-Å, respectively (Fig. 2c, d, Supplementary Figs.6and7,Table1).Themaprevealedanobviouselongatedextra densityaroundtheTM5ofFks1andFks2inthetransmembraneregion, whichalignswellwiththeEFUmolecule(Fig.2c,d).

In structures of both Fks1–EFU and Fks2–EFU, EFU was found stackingagainsttheTM5andembeddingintheexoplasmicleafletof thelipidbilayerinamannerofergosterol(Fig.2e,f).TheEFUmolecule locates~30Åfromtheactivesite,and~20Åfromtheputativeβ-1,3- glucan-transporting channel of Fks1 (Fig. 2g). The hydrophobic six- memberedringsaremainlystabilizedbyY638andF639inFks1(F657 andF658inFks2).Besides,thecarboxylicacidfunctionofEFUinter- actsdirectlywithE635andW695ofFks1(E654andW714ofFks2).In contrast,thehydrophilicglucoseandacetategroupofEFUhaslimited contact with FKS. Specifically, the glucose group is relatively more flexible according to its low density. It is consistent with previous findings that various EFU analogs with modification on these two groupsstillhighlyeffective36,37.

ToconfirmtheEFUbindingsite,weintroducedtwomutationsin thebindingsiteofFks1(E635AandF639S)andsubsequentlypurified themutantproteinsforaninvitroUDP-Gloglycosyltransferaseassay. We found that the enzymatic activities of E635A and F639S were unable to be inhibited by the EFU as the wild-type (WT) enzyme (Fig. 2h), indicating their essential role for EFU binding. To further evaluatethefunctionofkeyresiduesintheEFUbindingsite,wegen- eratedagroupofyeastfks1Δstrainswithplasmidcontainingwild-type Fks1ormutants,andmeasuredtheirMICvaluesofEFU(Fig.2i).Our resultsshowedE635A,F639S,F639A,andW695Lmutantsalldisplayed increasedMICvaluesat>30foldsofthatofwild-typeFks1,indicating theircrucialroleinEFUbinding;L642YandR645Fmutantsdisplayed increasedMICvaluesat2.5–5foldsofthatofwild-typeFks1,indicating theirmoderateroleinEFUbinding.Incontrast,thedetectedMICvalue of A631S, S634A, and F638A closely approximate that of wild-type, suggestingtheseresiduesmaynotbeimportantforEFU’sfunction.

Furthermore, we performed extensive molecular dynamics (MD) simulations of the wild type and mutant Fks1 in complex with enfumafungin embeddedinalipidbilayer(SupplementaryFig.8).Four EFU resistantmutationsE635A,F639A,F639S,andW695Lweretested, andtheRMSDvaluesofEFUcalculatedrelativetothestartingstruc- turewereusedtoexploreitsbindingstabilityintheactivesitesofFks1. The results revealed that the EFU is more dynamic in the mutants comparedtoWT,whilethebackbonesofallresiduesinbothWTand mutant Fks1 are stable. The differences indicate that these drug resistance mutant sites are crucial for EFU binding. A group of ibrexafungerp-resistant mutations has been identified in previous studies,includingF641,S645ofCandidaalbicansFKS1andE655,F659, S663,W715ofCandidaglabrataFKS2(SupplementaryTable2)32,34,38,39. Mostofthese residuesor thecorresponding residues in FKS1/2are located around the EFU molecule in Fks1–EFU and Fks2–EFU structures, further confirming our finding of the EFU binding site (Fig.2e,f,SupplementaryTable2).

Coincidentally,theEFUbindingsiteislargelyoverlappedwiththe echinocandin resistance “hot spot” regions (HS1: 635–649, HS2: 1354–1361, and HS3: 690–700)40,41. This is consistent with previous findingsthatsomemutationsareresistanttobothibrexafungerpand echinocandins,suchasF641,S645ofCandidaalbicansFKS1andE655, F659,W715ofCandidaglabrataFKS232,34.Ourresultalsoconfirmed that the mostfrequently observed echinocandin-resistant mutation, S643P,displayedincreasedMICvaluesofenfumafunginat~15foldsof thatofwild-typeFks1(Fig.2i).Meanwhile,duetoadifferentbinding mode,someechinocandin-resistantmutations,suchasR631,D632of CandidaglabrataFKS1,wereshowntobesensitivetoIbrexafungerp33.

RemodelingofthelipidenvironmentsurroundingFKSby Enfumafungin
TounderstandthemolecularmechanismbywhichEFUinhibitsFKS, wesuperimposedtheFks1/2structuresinEFUboundstateandapo state,andfoundtheiroverallstructuresarealmostidentical(Fig.3a). But compared to the apo structures, the phenyl groups of F639 in Fks1–EFU and F658 in Fks2–EFU rotate toward EFU molecule and clamptheEFUtogetherwiththephenylgroupsofY638inFks1and F657inFks2asavice(Fig.3b).Interestingly,movementofF639ofFks1 or F658 of Fks2 stabilizes a specific ordered lipid molecule in the groovebetweenTM5andTM8ofFks1/2-EFU,whichwasnotavailable intheapostateFks1/2(SupplementaryFig.9a).Thelipidisinparallelwith theEFUanddirectlyinteractswithmanyresiduesincludingF639of TM5,W695ofTM6,L1360andI1364ofTM8.Amongthem,F639and W695havebeenshowntoaffectthefunctionofEFU(Fig.2e).

Furthermore, a group of additional ordered densities corre- spondingtolipidswereidentifiedaroundTM8–IF3linkerandHH2Bin Fks1/2-EFUstructures,likelycausedbythepresenceofEFU(Fig.3c).To betterpresentthesedensities,wegeneratedrefined3DmapsofFks1 andFks2incomplexofEFUat3.3Åand3.4Åresolutionrespectively byfurther 3Dclassification andlocalrefinement(Fks1–EFU–Lip and Fks2–EFU–Lip)(SupplementaryFigs.6and7).Thelongoblateshapes oftheselipiddensitiessuggestthemtobesterols(Fig.3c).Remark- ably,twosterolmoleculesinterwindwitheachother,andstacktightly with W1773 of HH2B and Q1388 of TM8-IF3 linker (Fig. 3d).In vivo growth complementation assay of W1773A showed the mutant was unabletorescuefks1Δyeastgrowth,supportingitsessentialfunctional role(Fig.3f).Moreover,thesterolmoleculesalsointeractdirectlywith severalkeyEFUresistantmutantsin the Hot-Spot2,suchasF1366, F1370, and L1378 (Fig. 3d, e, Supplementary Table 2). It probably explainshowthemutantsinHotSpot2affecttheinhibitoryfunctionof antifungaldrugswhiletheyareatadistancefromtheEFUbindingsite.

Intactactivesiteandglucantransportchannelinthestructures ofFks1/2incomplexwithEFU
Previousstructuralstudiesrevealedthatthecentralcatalyticregionof Fks1adoptsacellulosesynthasefoldwithacytosolicconservedGT-A typeglycosyltransferasedomain23,24.However,severalessentialmotifs identified in cellulose synthase were either invisible or in different conformations in Fks1/2 structures, such as interface helix IF1/2/3, indicatingtheactivesiteofFKStobehighlydynamic(Fig.1,Supple- mentaryFig.1).Therefore,itisstillunclearwhethertheFKScatalyzes followingasimilarmechanismascellulosesynthases25,42,43.

Interestingly,well-orderedIFswereobservedinthestructuresof Fks1–EFU–Lip and Fks2–EFU–lip (Fig. 4a,Supplementary Fig. 9b). A conserved R–T/S–W motif (R443–T444–W445 in Fks1 and R462–S463–W464inFks2)andthesterolmoleculesmentionedabove playimportantroletostabilizeIF2andIF3(Fig.4a,b).TheR–T/S–W motif is located right before TMH1 (W445–Y469 in Fks1). The side chainsofT444andW445facetheactivesiteontherightintheapo statebutturnleftintheFks1–EFU–Lipstate,flippingbynearly180°. Conversely,thesidechainofW445facesleftwardintheapostatebut switchestotherightintheFks1–EFU–Lipstate.Specifically,R443of Fks1(R462inFks2)interactsdirectlywithD1416ofIF2andE1268ofIF3 bysaltbridges,whilethesidechainofW445andasterolmolecule interactwithL1414and L1417 ofIF2 byhydrophobic interactions. A horizontalsterolmoleculebelowthismotifandIF3furtherstabilizes thisarchitecturebyformingextensiveinteractions(Fig.4b).Notably, wefoundthatR443A,D1416A,andE1268Amutantswereallessential for Fks1’s function because they were unable to rescue fks1Δ yeast growthlikewild-typeFks1(Fig.4d).

SuperpositionofFks1/2–EFU–Lipandcellulosesynthaserevealed thatthepositionoftheirIFsandactivesiteswerequitesimilartoeach other,whiletheirputativeglucantransportchannelswereonlypar- tially similar (Supplementary Fig. 10a–i). Structure-based sequence alignmentalsoshowedlowsimilaritywithonlyafewconservedresi- dues identified,suchasthe magnesium coordinating residue D1102 andthephosphatecoordinatingresidueR1261ofFks1(Supplementary Fig.10j).Thisisconsistentwiththesequenceconservationanalysisof Fks1amongyeastandmajorpathogenicfungalspecies,whichshowed highconservationinGTbutrelativelylowerconservationintransport channel(SupplementaryFig.11).Theputativeglucantransportchan- nelofFKSweremainlycomposedofTM5-10andIF1-3.Similartothe structures of cellulose synthases, ordered IF1 and IF2 in Fks1/ 2–EFU–Lipconstitutetheentryofthechannel(Fig.4c).Ourprevious studieshaveperformedmutationsformostresiduesintheactivesite and confirmed their important roles23. Here, we introduced more mutations in IF2, IF3, and other residues in the glucan transport channel,andperformedthegrowthcomplementationassay(Fig.4d). TheresultsshowedmutationofkeyresiduesofIF2(F1258A,T1259A, andK1261A)inthechannelentryareessentialforFKS’sfunction,while most residues inside of the channel (E1402A, V1403A, Q1407A, Y1409A,S1410AofIF3,Q1305ofTM7)arerelativelylessimportant.

Although the corresponding regions ofIF3 and IF3–TM8 linker were identified as TM9–10 by inserting into the membrane in the previously reported structure of Fks1 in complex with glucan product24,thesetwoelementsinourstructuresarelocatedinasimilar position as many other membrane-integrated polysaccharide syn- thases,includingcellulosesynthase,chitinsynthase,andhyaluronan synthase25,44,45. The differences mayindicate the FKS has a dramatic conformational change during the glucan transport. The exporting pathofglucantoextracellularinFKSislikelythroughalateralopening, andmainlyformedbyTM8,TM10andIF3inourstructures(Fig.3d). Thegrowthcomplementationassaysformutationsofkeyresiduesin the exporting path (F1363, F1366 in TM8 and W1474 and S1478 in TM10)showedeachsinglemutantonlypartiallyaffectsFks1’sfunction, andmayworktogetherforglucantransporting(Fig.4d).Furthermore, because the TM8–IF3 linker and top parts of TM8 are amphipathic helices that half embedded in the membrane, the transmembrane regionadjacentTM8inourstructuresisonlyhalfasthickasnormal lipid bilayer, which favors the glucan exporting (Supplemen- taryFig.9b).

DiscoveryofanopenstateofFks1
Caspofungin(CPF)isthefirstmarketedechinocandinantifungalagent thatspecificallyinhibitsβ-1,3-glucansynthase46.Togaininsightsinto the inhibitory mechanism of echinocandin, we performed single- particlecryo-EManalysisonFks1incubatedwith2mMCPFovernight at4°C,andobtainedthecryo-EM3DmapofFks1inaresolutionof3.7- Å(SupplementaryFig.12).Unfortunately,noclearextradensitycor- respondingtoCPFwasidentifiedintheputativeechinocandinbinding site,whichwasproposedbasedonechinocandin-resistantmutations, indicatingthatthestructureremainsinanapostate.Interestingly,this structureshowedamoreopenactivesite(namedFks1–open),whose architecturediffersfromthatofFks1ineithertheapoorEFU-bound states(Fig.5a).

StructuralalignmentbetweenFks1–openandFks1–aporevealed dramaticconformationalchangesinbothTMD2andtheIF1–3(Fig.5a). SomeimportantresiduesintheactivesiteofGTDwerealsoshiftedin Fks1–open,suchasN1104,E1155,H1195,K1246,andR1248.Compared totheFks1–apo,thewholeTMD2inFks1–openrotatedandmovedby ~5Å in relative to TMD1, resulting in a more open glucantransport channelbetweenTMD1andTMD2;IF1andTM8–IF3linkerrotate~90° towardoutside,whileIF3isshiftedby~20Åaccordingly,generatinga more open active site (Fig. 5b, c). Therefore, we named the con- formation of Fks1/2–apo and Fks1/2–EFU the “basal state” as they resemble other polysaccharide synthases, such as cellulose synthase25,44,45. Infact,wehavefurtherprocessedacryo-EMdatasetof Fks1intheapostatebyfocused3DclassificationincryoSPARCandare able to separate a small subset of particles (9.8%) adopting a con- formationliketheopenstate(SupplementaryFig.13).Thissuggests thatFks1inboth the basalandopen state areavailable insolution. Whilethebasalstateaccountsforthemajority,theovernightincuba- tionofFks1probablyfacilitatestheconformationalchangefromthe basaltotheopenstate.BecausethesemovedelementsinFks1–open have been shown to be directly involved in active site or glucan transporting, the conformationalchange of Fks1 between basal and openstatesisclearlylinkedtoitsfunction.

ImplicationsfortwodifferentstatesofFKS
Interestingly,thestructureofFks1–openissimilarwiththereported structures of Fks1 that was purified in glyco-diosgenin (GDN) (Sup- plementaryFig.1).AlthoughtheirTMD2areindifferentpositions,IF1/ 3,partofTM8,andTM8–IF3linkerofthesestructuresarealllocating outofthecoredomainandthewholestructuresareintheopenstate (Supplementary Fig. 1). Investigation on all available FKS structures showedthatFks1/2inbasalstatewascapturedinbuffercontaining LMNG/CHS, while the open state was captured in buffer containing GDN. As a cholesterol analog, CHS closely mimics the property of cholesterol/ergosterolinnativemembranes,andiscommonlyusedto maintain the native structure of membrane protein during purification47,48.Wenoticedthatasterolmoleculewasidentifiedunder TM8–IF3 linker of Fks1 in the basal state, but disappeared in the structureofFks1intheopenstate(Fig.5c).Thesterolinteractsdirectly withTM8,TM10andTM8–IF3linker,andlikelyplaysanimportantrole instabilizingtheFKSinthebasalstate.Overnightplacementormiss- ingCHSinthesolutionmayreleasethesterolfromthesiteandthus induce the conformational change from the basal state to the openstate.

Notably, although the FKS purified inLMNG-CHS or GDN were capturedindifferentstates,theproteininbothdetergentswasproved tobeactive23,24.Therefore,thebasalandopenstatesofFks1areboth likelytobeintermediatestatesthatoccurduring catalysis.In other words, a flexible and dynamic active site identified in the above structuresmaybeessentialforthecatalysisofFKS.Toconfirmthis hypothesis,wegeneratedtwoFks1doublemutants:A1264C–D1416C andS1415C–A1485C,whicharesupposedtofixFks1inbasaloropen architectures,respectively,byformingadisulfidebond(Fig.5b,c).In vivo growth complementation assays of the Fks1 double mutants showedthatneitherofthemwasabletorescuefks1Δyeastgrowthon plates containing FK506 like the single mutants or wild type Fks1 (Fig.5d).WealsopurifiedA1264C–D1416CandS1415C–A1485C,and measured their in vitro activities (Fig. 5e). The results showed the enzymatic activities of both double mutants were dramatically reducedwhencomparedwithwildtypeFks1.Takentogether,these resultssupportthatbothbasalandopenstatesofFks1areimportant foritsfunction.

Methods
ExpressionandpurificationofendogenousFks1andFks2
The C-terminal of FKS1 in the yeast strain W303 (EUROSCARF) was engineeredwithatriple-FLAGandSpHis5usingaPCR-basedgenomic epitope-tagging method. Using the same method, FKS2 was engi- neeredinanFKS1knockoutW303strain.Theyeaststrainswerecul- turedinYPDmedium(10g/Lyeastextract,20g/Lpeptone,and20g/L glucose)forapproximately20hbeforeharvesting.Cellpelletswere resuspendedinlysisbuffercontaining20mMTris-HCl(pH7.4),0.2M sorbitol, 50mM potassium acetate, 2mM EDTA, and 1mM phe- nylmethylsulfonyl fluoride (PMSF). Cells were lysed using a French press at 15,000psi. The lysate was subjected to centrifugation at 10,000×g for 30min at 4°C, and the supernatant was further cen- trifugedat100,000×gfor60minat4°C.Themembranepelletwas collected and resuspended in buffer A, which consisted of 10% gly- cerol, 20mM Tris-HCl (pH 7.4), 1% n-dodecyl-β-D-maltoside (DDM), 0.1% cholesteryl hydrogen succinate (CHS), 500mM NaCl, 1mM MgCl , 1mM EDTA, and 1mM PMSF. After incubation at 4°C for 30min, the sample was centrifuged at 100,000×g for 30min to remove insoluble material. The solubilized membrane fraction was appliedtoapre-equilibratedanti-FLAGM2affinitycolumn(GenScript) at4°C.AfterextensivewashingwithbufferB(20mMHEPES,pH7.4, 150mMNaCl,0.01%laurylmaltoseneopentylglycol(LMNG),0.001% CHS,and1mMMgCl2),theboundproteinswereelutedusingbufferB supplementedwith0.15mg/mL3×FLAGpeptide.Theelutedfraction was further purified via size-exclusion chromatography using a Superose610/300IncreasecolumnequilibratedinbufferC(20mM HEPES,pH7.4,150mMNaCl,0.003%LMNG,0.0003%CHS,and1mM MgCl2).Thepurityofthefinalproteinpreparationwasassessedby SDS-PAGE, and the protein was concentrated for subsequent cryo- electronmicroscopy(cryo-EM)analysis.

ExpressionandpurificationofRho1
The cDNA encoding RHO1 was amplified from the genome of Sac- charomycescerevisiaestrainW303andsubclonedintothepFastBac vector with an N-terminal 6×His tag. A mutation (Q68H) was intro- duced to generate an active form of Rho1 using a PCR-based site- directedmutagenesisstrategy.TheBac-to-Bacbaculovirusexpression system in Spodoptera frugiperda cells (Gibco Sf9 cells, cat. no. 11496015)wasusedforproteinexpression.SF9cellswereinfectedata densityof2×106cells/mLandharvestedat60hpost-infection.Col- lected cells were resuspended in Buffer A (20mM Tris-HCl, pH 7.4, 500mM NaCl, 10% glycerol, and 1mM MgCl₂) and lysed on ice by sonication.Celldebriswasremovedbycentrifugationat15,000×gfor 30minat4°C,andthesupernatantwasappliedtoa2mLNickel–NTA column.Thecolumnwaswashedwithbuffercontaining20mMHEPES (pH7.4),20mMimidazole,and150mMNaCl,andtheboundprotein waselutedusingthewashbuffersupplementedwith200mMimida- zole.Theelutedproteinwasconcentratedandfurtherpurifiedbysize- exclusionchromatographyusingaSuperose610/300GLcolumn(GE Healthcare). Peak fractions were collected and used forsubsequent functionalassays.

Cryo-electronmicroscopy
Aliquots(2.5μL)ofFks1andFks2aloneorincomplexwithEFUata concentration of approximately 3mg/mL were applied to glow- discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh) and flash-frozen inliquid ethane using a FEI Vitrobot Mark IV. Grid screeningwasperformedona300-kVFEITF30electronmicroscope, andCryo-EMdatawereautomaticallycollectedusingEPUoneithera 200-kVThermoFisherScientificGlacios2ora300-kVThermoFisher ScientificKriosG4electronmicroscope.Dataacquisitionwascarried outwithdefocusvaluesrangingfrom−1.0to−2.0μm.Themicroscope wasoperatedwithaK3directelectrondetectoratanominalmagni- ficationof130,000×.Thetotalelectrondosewassetto40–60elec- tronsperÅ²atthesamplelevel.

Cryo-EMimageprocessing
AlldataprocessingstepswereperformedusingCryoSPARC.Thecol- lectedmoviesweremotion-corrected,andcontrasttransferfunction (CTF) parameters were estimated. Micrographs were then filtered using exposure curation. Particle picking was conducted with a template-based picker usingmanuallyselected 2Dclassaveragesas templates,followedbyparticleextraction.After2Dclassificationand Ab-initioreconstruction,selectedparticleswerefurtherrefinedusing non-uniformrefinement.

Forthestructuredeterminationofapo-stateFks1,atotalof6816 raw movie micrographs were collected. Automated particle picking yielded 401,620 particles,which weresubjected to 2Dclassification and heterogeneous refinement. Based on the quality assessment of three heterogeneously refined 3D maps, 248,992 particles were selected for subsequent refinement and post-processing. The final reconstruction, performed with C1 symmetry, resulted in a 3.23Å resolution3Dmap.

ForthestructuredeterminationofEFU-boundstateFks1,atotalof 6457 raw movie micrographs were collected. Automated particle pickingyielded479,938particles,whichweresubjectedto2Dclassi- ficationandheterogeneousrefinement.Basedonthequalityassess- ment of three heterogeneously refined 3D maps, 301,005 particles were selected for subsequent refinement and post-processing. The finalreconstruction,performedwithC1symmetry,resultedina2.97Å resolution3Dmap.Afterfurther3Dclassificationandlocalrefinement, improved 3D maps of Fks1with ordered IFs were obtained at 3.3Å resolution(Fks1–EFU–Lip).

Forthestructuredeterminationofopen-stateFks1,atotalof4402 raw movie micrographs were collected. Automated particle picking yielded 616,794 particles, which were subjected to 2D classification and heterogeneous refinement. Based on the quality assessment of threeheterogeneouslyrefined3Dmaps,142,777particleswereselec- tedforsubsequentrefinementandpost-processing.Thefinalrecon- struction, performed with C1 symmetry, resulted in a 3.72Å resolution3Dmap.

ForthestructuredeterminationofapostateFks2,atotalof6588 raw movie micrographs were collected. Automated particle picking yielded621,453particles,whichweresubjectedto2Dclassificationand heterogeneousrefinement.Basedonthequalityassessmentofthree heterogeneouslyrefined3Dmaps,97,369particleswereselectedfor subsequentrefinementandpost-processing.Thefinalreconstruction, performedwithC1symmetry,resultedina3.64Åresolution3Dmap.

ForthestructuredeterminationofEFU-stateFks2,atotalof9020 raw movie micrographs were collected. Automated particle picking yielded510,461particles,whichweresubjectedto2Dclassificationand heterogeneousrefinement.Basedonthequalityassessmentofthree heterogeneouslyrefined3Dmaps,250,126particleswereselectedfor subsequentrefinementandpost-processing.Thefinalreconstruction, performedwithC1symmetry,resultedina3.32Åresolution3Dmap. Afterfurther3Dclassificationandlocalrefinement,improved3Dmaps of Fks2 with ordered IFs were obtained at 3.4Å resolution (Fks2–EFU–Lip).

Structuralmodeling,refinement,andvalidation
ThepredictedstructuresofFks1andFks2,generatedbyAlphaFold2, wereusedasinitialmodels.Thesemodelswerefittedintothecryo-EM densitymapsandmanuallyadjustedusingCOOTandChimera.The complete Fks1 and Fks2 models were refined through real-space refinement in the PHENIX program, followed by further manual adjustments in COOT. Model validation was performed using Mol- Probity.StructuralfigureswerepreparedusingChimeraandPyMOL (https://pymol.org/2/).

UDP-Gloglycosyltransferaseassay
The enzymatic activity of wild-type (WT) FKS and its mutants was assessed using the UDP-Glo™ Glycosyltransferase Assay (Promega, Catalog No. V6961), which quantifies the activity of glycosyl- transferases utilizing UDP-sugars as substrates. Each reaction was performed in a total volume of 5μL, containing 0.12μg of purified proteins,20mMHEPES(pH7.4),150mMNaCl,0.025%n-dodecyl-β-D- maltoside (DDM), 0.0025% cholesteryl hydrogen succinate (CHS), 1mMMgCl ,1mMGTP-γS,1mMUDP-glucose,and1mMglucose.The 2 reactionmixtureswereincubatedat30°Cfor30min,followedbythe additionof5μLUDPDetectionReagent.Afteranadditional60min incubationatroomtemperature,luminescencewasmeasuredusinga SynergyH1HybridMulti-ModeMicroplateReader(BioTek).

Colonygrowthassay
We prepared the Fks1 knockout strain (ΔFks1) in the W303 strain (EUROSCARF).Fks1mutantsandtruncationswereconstructedusing plasmid pRS423–His3 (Addgene) and transformed into the ΔFks1 strain.Wild-typeyeaststrainsandFKS1knockoutstrainswereinitially culturedinSDmediumat30°Covernighttoreachthesameoptical density(OD).Serial10-folddilutionsofthecellswerethenprepared andspottedontoSD-Hisplateswithorwithout0.1μg/mLFK506.The plateswereincubatedat30°Cfor2days,afterwhichcolonygrowth wasassessed.

Antifungalsusceptibilitytesting
Antifungalagentswerepreparedasstocksolutionsataconcentration of 10mg/mL in dimethyl sulfoxide (DMSO) and stored at −20°C, exceptforcaspofungin,whichwasdissolvedinwaterduetoitshigh solubility.Thefinalconcentrationsusedintheassayswereasindicated in the text and figures. For micro-culture assays involving multiple samples, late logarithmic-phase cultures grown in SD-His medium weredilutedtoafinalcelldensityof2×106cells/mLin200μLofSD- Hismediumcontainingseriallyincreasingconcentrationsoftheanti- fungalagentsoranequivalentvolumeofsolventasacontrol.Cultures wereincubatedat30°Cwithcontinuousagitationonanorbitalroller, andtheturbiditywasmeasuredfollowing24and48hofincubation. The minimum inhibitory concentration (MIC ) was defined as the 50 lowestantifungalconcentrationrequiredtoinhibit50%ofmicrobial growthafter24hofincubation.Allreportedvaluesrepresentthemean ofatleastthreeindependentexperiments.

Moleculardynamicssimulations
All-atommoleculardynamics(MD)simulations,wereperformedusing GROMACS2025.1withtheCHARMM36mforcefieldfortheprotein andCGenFFparametersfortheligand.Thesimulationsystemswere constructed using CHARMM-GUI, embedding the protein-ligand complex in a POPC bilayer solvated with TIP3P water and 0.15M NaCl; detailed system parameters are provided in Supplementary Table3.Followingenergyminimization,thesystemswereequilibrated using the standard CHARMM-GUI six-step protocol with gradually releasedpositionrestraints.Fourindependent100nsproductionruns wereperformedforeachsystemwitha2fstimestep.Thetemperature and pressure were maintained at 303.15K and 1.0bar using the V-rescalethermostatandC-rescalebarostat,respectively,whilelong- rangeelectrostaticinteractionsweretreatedusingthePMEmethod andhydrogenbondswereconstrainedusingtheLINCSalgorithm.

Structurepredictionandanalysis
Structuralcomparisonsoftheapo-Fks1(SupplementaryFig.1)utilized thepredictedmodelofS.cerevisiaeFks1retrievedfromtheAlphaFold ProteinStructureDatabase(https://alphafold.ebi.ac.uk;EntryID:AF- P38631-F1,modelversion4).Fortheprotein-ligandcomplexpredic- tions (Supplementary Fig. 14), the structures of S. cerevisiae Fks1 (UniProtID:P38631)andC.albicansFks1(UniProtID:A0A1D8PCT0)in complex with enfumafungin (EFU) were generated using a locally deployedversionofAlphaFold3.Thepredictionswereperformedin April2025.TheSMILESstringofenfumafunginwasprovidedasthe ligandinput.Theconfidenceofthepredictedmodelswasevaluated using the predicted localdistancedifference test(pLDDT) for local residue quality and the predicted aligned error (PAE) for domain packingandligandplacementreliability.Visualizationsofthemodels coloredbypLDDTscoresandthecorrespondingPAEplotsarepro- videdinSupplementaryFig.14.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript written by You et al. describes the structure determination of cryo-EM structures of two beta-1,3-glucan synthases, Fks1 and Fks2 from Saccharomyces cerevisiae.\nThe Fks2 structure was first reported here. However, there is no evidence for the “functions using a different mechanism from Fks1” (P3L80). The authors suggested that the regulatory subunit of Fks2 can be Rho3, not Rho1, but this is also just a speculation only on an in vitro UDP-Glo GT assay (Fig. 1a).\nThe structures of Fks1 have been previously reported by at least three papers, and its molecular mechanism of glucan synthesis is already known. The authors found a novel enufumafungin (EFU) binding site that cannot be predicted by AlphaFold3 (Supp. Fig. 13). The authors conducted both in vitro and in vivo assays of the mutants in this area. Therefore, the EFU binding site identified in this study is probably correct. The observation of IF1-3 helices, which play important roles in its synthase function, was also interesting and informative.\nBecause no EM map for the putative caspofungin (CPF) binding site is shown (P9L253-257), the discussion about the possible conformational fixing to the open state (Figs 5 and 6) cannot be supported.\nI found many grammatical errors and unusual word usages in this paper.\nMajor points:\n1. P2L46 and P8L235. 4-glucose glucan (?) Do you mean a laminaritetraose?\n2. P3L79. Supplementary Figure 2a-c does not match the text.\n3. P4L106. TM9, TM14, and HH2A are shown in Fig. 1e.\n4. P5L117. “hemiacetal function and carboxylic acid function” function? Do you mean moiety or group?\n5. Fig. 2a. The glucose unit is not glucose (no -CH2-OH group of C6).\n6. P7L182. “TM8-IF3 linker”; P8L205. “well ordered Ifs”. I know TM means transmembrane helix, but I am not familiar with the abbreviation “IF”.\n7. P8L205. R-T/S-W motif seems to correspond to R433-T44-W445 in Fks1. Is this correct?\n8. P8L208-209. “The R-T/S-W motif locates adjacent before TMH1, and its side chains are all flipped nearly 180° from the Ifs disordered state to ordered state.” I could not understand this sentence when I saw Fig. 4a, b.\n9. P8L208. TMH1 =Transmembrane helix 1?\n10. P8L214. “R443L” In Fig. 4b, there is only R443A.\nMinor points:\n1. P2L54. naturally triterpene -> natural triterpene\n2. P3L69 C-terminal of FKS1 or FKS2 -> C-terminus ? FKS1 and FKS2 are written in italics and seem to be gene names, but “C-terminus/C-terminal” is only applicable for polysaccharides (proteins).\n3. P3L73. .. and the lauryl -> and lauryl\n4. P3L73-74. .. (LMNG) and CHS were used ..\n5. P3L79. .. activity in (under?) the same condition\n6. P3L80. .. may function using ..\n7. P8L211. “ion bonds” salt bridges?\n8. P8L221. .. but relatively lower ..\n9. P9L238. .. may indicate the ..\n10. P8L249-250. .. that specifically inhibits ..\n11. P8L255. basing on -> based on?", "reviewer_2": "A hallmark of successful clinical management for invasive fungal disease is the availability of effective antifungal therapy, and glucan synthase inhibitors represent one of the most important drug classes. The echinocandins, approved for clinical use nearly 25 years ago, remain first-line therapy for a broad range of candidiasis and candidemia. Although they are limited to intravenous administration, they have recently been joined by the first-generation orally bioavailable enfumafungin derivatives, which also inhibit glucan synthase. Genetic studies of resistance suggest that the enfumafungin drug binds to the glucan synthase complex at a distinct yet partially overlapping site compared with the echinocandins.\nExtensive knowledge has accumulated regarding the genetics, resistance mechanisms, and biochemical and kinetic properties of glucan synthase, encoded by the FKS genes. However, until recently, little was known about the enzyme’s structural organization or the molecular basis of drug–target interactions underlying inhibition—information critical for optimizing antifungal therapy. Recent cryo-EM studies from this group and others have begun to illuminate these structural features, especially concerning resistance-associated amino acid substitutions. Yet, our understanding of how echinocandins and related enfumafungin drugs achieve target inhibition remains largely inferential, based primarily on genetic analyses of resistance.\nThis new study by a leading glucan synthase structural biology group provides much-needed insight into the structural basis of drug–target interactions responsible for enzyme inhibition. The authors resolved five cryo-EM structures of Fks1 and Fks2 in the apo state, in complex with the triterpenoid enfumafungin drug, as well as in an open state induced by echinocandin drug caspofungin. Structural analysis along with strategic mutagenesis revealed the putative enfumafungin binding site, and the likely mechanism for clinically important drug-resistant mutations. Consistent with inferences from numerous genetic studies in the literature and some new mutant analysis, it is proposed that the enfumafungin attaches to transmembrane helix TM5 of the glucan synthase, reorganizes its nearby lipid environment, and stabilizes the enzyme in a unique basal state with intact active site. New insights about the location of the glucan channel, which appears to lie between TMDs 1 and 2 are exciting.\nThe elucidation of a structure from cryoEM studies with the enfumafungin bound is a key advancement of this work. Furthermore, the observation of an elongated extra density around the TM5 of Fks1 and Fks2 in the transmembrane region, which aligned well with the EFU molecule, was a major finding.\nOverall, this is an important contribution that strongly advances the field, and supports more advanced computationally assisted drug design to develop next generation drugs that\noptimize enzyme inhibition across a range of high-threat fungi and have the potential to overcome FKS hot-spot resistance known to cause refractory drug responses in patients.\nRevision Considerations\nThe manuscript would benefit from some minor English editing:\nLine 194: Hot spot 2 effect the inhibitoty function. Please correct to: Hot Spot 2 affect inhibitory function\nLine 203: Should be: Therefore, it’s still unclear whether the FKS catalyze following a similar …\nLine 79. ‘It indicates Fks2 may functions using a different mechanism from Fks1.’ It is well-known that FKS1 and FKS2 have different regulation and potential functon(e.g., Healey et al. 2020; Katiyar et al. 2012; Garcia-Effron et al. 2009; Gonzalez-Jimenez et al, 2025; Suwunnakorn et al. 2018)\nLine 83. It suggests the regulatory subunit of Fks2 is probably Rho3. This is a helpful and insightful observation. Do we understand how Rho activates from a structural perspective?\nIt was reassuring to see that the enfumafungin binding site was largely overlapped with the echinocandin resistance “hot spot” regions, which aligns very well with published genetic studies, as indicated.\nLines 191-195. The potential role of a dual sterols in interacting with enfumafungin and drug resistant mutants is intriguing. However, azoles which block sterol biosynthesis and levels in the membrane are not known to have antagonistic nor synergistic effects on echinocandin nor enfumafungin action. The authors may want to speculate about this known observation.\nLine 255. Did the authors look to see if caspofungin could displace or compete in enfumafungin bound density observed from the enfumafungin bound? This would get partially at the point of an overlapping binding site.\nThe general Discussion is sparse and should be expanded to discuss opportunities and alignment with genetic and biochemical studies. It would benefit from a description of study limitations. It is important note that in general structural resolution of 2.5–3.0 Å allows side chains to be visualized, but hydrogen positions are uncertain. For some structures at or above 3.5 Å, only backbone and bulky side chains are clear, while any atomic interactions must be inferred.\nAlso, the structural studies were conducted on the Saccharomyces cerevisiae glucan synthase, which is sensitive to enfumafungin but weakly sensitive to echinocandins. The authors did attempt to affirm the structural binding motifs with relevant mutant enzymes from clinically significant pathogenic Candida species, but it would be valuable to understand potentials gaps in the analysis. This particularly important in terms of interactions with sterol, bound lipids and impact on enzyme inhibition and resistance. While alpha-fold may be of limited value here, what about other computational modeling approaches? Were any energy minimizations run with bound drug for wild-type and mutant enzymes?", "reviewer_3": "In this work, You and co-workers provide cryoEM structures of β-1,3-glucan synthases Fks1 and Fks2 in the apo state, the triterpenoid drug enfumafungin-bound state, and an open state induced by echinocandin incubation. It is worth mentioning that structures of Fsk1 were previously reported (Li et al. Cryo-EM structure of the β-1,3-glucan synthase FKS1-Rho1 complex. Nat Commun. 16:2054 (2025); Zhao et al. Structure of a fungal 1,3-β-glucan synthase. Sci Adv. 9:eadh7820 (2023); Hu et al. Structural and mechanistic insights into fungal β-1,3-glucan synthase FKS1. Nature 616(7955):190-198 (2023). In combination with single-point mutagenesis, activity measurements, growth complementation and antifungal susceptibility assays, the authors provide new and important insights on the molecular basis of catalytic cycle, activation by Rho1, and inhibition by triterpenoid and echinocandins of β-1,3-glucan synthases. The article is well written and the interpretation of the experimental data support the proposed model. Therefore, I support publication of the manuscript in Nature Communications.\nSome suggestions for improvement are listed below:\n- page 2, line 38: I would suggest to replacing ““membrane bound enzyme” by “integral membrane enzyme”.\n- page 2, line 39: “catalyzing the synthesis of β-1,3-glucan”. Please, incorporate the reaction scheme as Figure 1a.\n- page 2, line 40: “Fungal β-1,3-glucan synthase is primarily encoded by FKS1 or FKS27, the activity of which was regulated by Rho1”. Please, elaborate the main similarities/differences and how Rho1 regulates the enzymes.\n- page 2, line 46: “in presence of a 4-glucose glucan have been determined”. Please, revise and incorporate the precise identity/nomenclature.\n- page 2, line 47: Please, incorporate FKS1 and FKS2 belongs to CAZy family GT48. It would be interesting the authors could elaborate - and also include a sequence-based structure alignment, highlighting important residues involved in catalysis/beta-glucan transport and protein-protein/protein-inhibitors interactions - on the homology of FSK1/FSK2 with other enzymes adopting a conserved GT-A type glycosyltransferase domain and a transmembrane glycan-transporting channel - e.g. cellulose synthase, chitin synthase and hyaluronan synthase.\n- page 3, line 76: “Functional characterization with in vitro UDP-Glo glycosyltransferase assay”. Please, confirm the use of UDP-glucose as sugar donor in the Methods section.\n- page 4, line 91: “The overall structures of Fks1-apo and Fks2-apo are similar to each other with a root-mean-square-deviation (RMSD) of ~1.5 Å over 1449 residues”. Please, state UniProt codes and total number of residues for both enzymes.\n- page 4, line 100: “… a glycosyltransferase (GT) domain inserted between TM6 and TM7…” A good opportunity to introduce a topology model on Fks1/2 as Figure 1f.\n- page 5, line 128: “EFU was found stacking against the TM5 and embedding in the exoplasmic leaflet of the lipid bilayer”. Please, could the authors state how far is the EFU binding site from the beta-glucan-transporting channel - entrance/exit - of Fks1 and Rho1? Please, incorporate an additional panel in Figure 2 and/or Figure 4, including the β-1,3-glucan synthase in surface representation - e.g. use PDB 7YUY and 8WLA as references - different orientations - cytosolic/extracellular entrances - to better show the channel, and secondary structures involved.\nPlease, also check:\n- page 1, line 25: please replace “enviroment” by “environment”.\n- page 2, line 44: please homogenize FKS1 and FKS2 or Fks1 and Fks2, when appropriate.\n- page 7, line 194: please replace “inhibitoty” by “inhibitory”."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript written by You et al. describes the structure determination of\ncryo-EM structures of two beta-1,3-glucan synthases, Fks1 and Fks2 from\nSaccharomyces cerevisiae.\nThe Fks2 structure was first reported here. However, there is no evidence for the\n“functions using a different mechanism from Fks1” (P3L80). The authors\nsuggested that the regulatory subunit of Fks2 can be Rho3, not Rho1, but this is\nalso just a speculation only on an in vitro UDP-Glo GT assay (Fig. 1a).\nThanks for the valuable comment.\nWe totally agree with the reviewer that the statements are of insufficient evidence.\nAs the reviewer pointed out, the speculation is just only on the in vitro UDP-Glo\nassay which didn’t show activation of Fks2 by Rho1. Although Rho3 was detected\nin the pull down proteome of purified Fks2 with cell lysate, very few peptides were\nidentified and thus the evidence is not strong enough to make conclusion.\nTo address this question, we firstly tried to capture the complex of Fks2 and\nRho1/3. Because the purified endogenous Fks1 sample didn’t include detectable\nRho1 or Rho3, indicating binding of the two protein is weak or dynamic. To capture\nthe complex, we incubated purified Fks2 and Rho1/Rho3 with crosslinker BS3, and\nthen separated the crosslinked complex using size exclusion column.To our\nsurprise, the shifted position of gel filtration peak indicated the complex of Fks2-\nRho1 was successfully purified, while the Fks2 and Rho3 did not form complex\n(Supplemental Fig. 3a, Response Figure 1a, b). We further performed EM analysis\non crosslinked Fks2-Rho1 complex, and the 2D class averages clearly showed\nadditional density corresponding to Rho1 (Supplemental Fig. 3b, Response Figure\n1c). The detected binding position of Rho1 is consistent with published structure\nof Fks1-Rho1 complex. Together, these results support that Rho1 is the regulatory\nsubunit for Fks2.\nThen we looked into why no activity of Fks2 with Rho1 was orginally detected in\nthe in vitro UDP-Glo assay, and noticed that the constitutively active Rho1-Q68H\nmutant was used in earlier experiments but the wild type Rho1 was used in\nexperiments during the revision. We speculated that the Rho1-Q68H mutant is\nprobably unable to activate Fks2 like to Fks1. Moreover, we found Fks2 did not\nform complex with Rho1-Q68H like with Rho1 after crosslinking in size-exclusion\nchromatography (Response Figure 1d). Therefore, we redid the in vitro UDP-Glo\nglycosyltransferase assay with Fks2 and wild type Rho1 in presence of GTP-rS, and\nindeed detected glycosyltransferase activity of Fks2 as high as Fks1 (Fig. 1b,\nResponse Figure 1e). This result further confirmed that the essential role of Rho1\nfor Fks2.\nAs for why the constitutively active Rho1-Q68H mutant is unable to activate Fks2\nlike to Fks1, we analyzed the potential interfaces between Rho1 and Fks1/2, and\nnoted that the residues adjacent to Q68 of Rho1 in Fks1 and Fks2 are not exactly\nconserved (Response Figure 1f). Specifically, a nearby 377FVKR380 motif in Fks1\nis equivalence of 396YVKS399 in Fks2, the difference of which may affect their\ninteraction with Q68H of Rho1 and cause the problem we encountered. We also .\nOf course, more experiments need to be done to fully unveil this question. In\naddition, whether Rho3 is also the regulatory subunit of Fks2 in vivo still await\nmore studies. As these two questions are not the major point in this manuscript,\nwe decided to not include them here and hope to fully reveal the questions in\nfuture.\nIn light of these new results, we have removed earlier statements about Fks2 in\nthe revised manuscript, and concluded that Rho1 is the regulatory subunit for both\nFks1 and Fks2.\nResponse Fig. 1 Interactions between Fks2 and Rho1 or Rho3. a Gel filtration\nprofiles of Fks2 alone, and the mixture of crosslinker BS3, Fks2 and Rho1 in\npresence of GTPγS. Peak corresponding to the Fks2-Rho1 complex was observed.\nb Gel filtration profile of the mixture of crosslinker BS3, Fks2 and Rho3. Fks2-\nRho3 complex was not detected. c 2D class averages of Fks2 alone and crosslinked\nFks2-Rho1 complex. The additional densities corresponding to Rho1 were\nhighlighted by red arrows.d Gel filtration profile of the mixture of crosslinker BS3,\nFks2 and Rho1-Q68H. Fks2-Rho1-Q68H complex was not detected.e In vitro\nUDP-Glo glycosyltransferase assay with purified Fks2 with Rho1 in presence of\nGTPγS or Rho1-Q68H. The reaction without FKS was used as the control.f\nInterfaces between Rho1 and Fks1/2. Key residues involved in the interaction are\nshown as sticks.\nThe structures of Fks1 have been previously reported by at least three papers, and\nits molecular mechanism of glucan synthesis is already known. The authors found\na novel enufumafungin (EFU) binding site that cannot be predicted by AlphaFold3\n(Supp. Fig. 13). The authors conducted both in vitro and in vivo assays of the\nmutants in this area. Therefore, the EFU binding site identified in this study is\nprobably correct. The observation of IF1-3 helices, which play important roles in\nits synthase function, was also interesting and informative.\nWe thank the reviewer for the positive comments of our work.\nBecause no EM map for the putative caspofungin (CPF) binding site is shown\n(P9L253-257), the discussion about the possible conformational fixing to the\nopen state (Figs 5 and 6) cannot be supported.\nThanks for the valuable comment.\nWe totally agree with the reviewer that, in the absence of an EM density of\ncaspofungin (CPF), the discussion about CPF incubation-driven conformational\nfixing is likely of insufficient evidence and inappropriate. Therefore, in the revised\nmanuscript, we explicitly claim the captured structure in open conformation is\nlikely not caused by CPF incubation and related to the CPF binding. Instead, we\nspeculate the open state is probably caused by the process of overnight incubation,\nIn fact, we have further processed the cryo-EM dataset of Fks1 in apo state by\nfocused 3D classification in cryoSPARC and are able to separate a small subset\nparticles (9.8%) adopting the conformation alike open state (Supplementary Fig.\n13). This suggests that Fks1 in both the basal and open state are available in\nsolution, although the basal state accounts for the majority.The overnight\nincubation of Fks1 probably facilitates the conformational change from basal to\nopen state. Accordingly, we have revised the text and Figures in the manuscript to\nclarify the interpretion of the Fks1-open structure. We hope these changes can\naddress the concerns and align the interpretation strictly with the structural\nevidence in hand.\nI found many grammatical errors and unusual word usages in this paper.\nMajor points:\n1. P2L46 and P8L235. 4-glucose glucan (?) Do you mean a laminaritetraose?\nThanks for the comment. The “4-glucose glucan” was used following the original\ncited paper (ref.24). Because the structure was determined using sample incubated\nwith UDP-Glc, authors proposed it to be a product-bound state of the enzyme.\nThe glucan was modeled basing on the density, which fits well with 4-glucoses.\nTo be more clear, we revised the “4-glucose glucan” to “glucan product” in these\ntwo sentences.\n2. P3L79. Supplementary Figure 2a-c does not match the text.\nWe thank the reviewer for the comment and apologize for our careless. The\ncitation has been corrected to “Supplementary Figure 2a–b” in the revised\nmanuscript.\n3. P4L106. TM9, TM14, and HH2A are shown in Fig. 1e.\nWe thank the reviewer for the comment and apologize for our careless. We have\npresented and labeled the TM9, TM14, and HH2A in Fig. 1e.\n4. P5L117. “hemiacetal function and carboxylic acid function” function? Do you\nmean moiety or group?\nThanks for the comment. We have revised it to “hemiacetal moiety and carboxylic\nacid group (Fig. 2a).”\n5. Fig. 2a. The glucose unit is not glucose (no -CH2-OH group of C6).\nWe thank the reviewer for the comment and apologize for the mistake. We have\nadd the missing -CH2-OH group of C6 in the Fig.2a.\n6. P7L182. “TM8-IF3 linker”; P8L205. “well ordered Ifs”. I know TM means\ntransmembrane helix, but I am not familiar with the abbreviation “IF”.\nThanks for the comment. “IFs” refers to interface helices, which were originally\ncharacterized in cellulose synthase as conserved elements in the active site. To\nimprove clarity, we have added this definition in the second paragraph as\nfollows:“such as the conserved horizontal interface helices (IF1/2/3) that were\noriginally characterized in cellulose synthase”\n7. P8L205. R-T/S-W motif seems to correspond to R433-T444-W445 in Fks1. Is\nthis correct?\nThanks for the comment. The R-T/S-W motif correspond to R443-T444-W445 in\nFks1 and R462-S463-W464 in Fks2.To improve clarity, we have added the\nstatement in the revised manuscript as follows: “A conserved R-T/S-W motif\n(R443-T444-W445 in Fks1 and R462-S463-W464 in Fks2) and the sterol\nmolecules mentioned above play important role to stabilize IF2 and IF3”.\n8. P8L208-209. “The R-T/S-W motif locates adjacent before TMH1, and its side\nchains are all flipped nearly 180° from the Ifs disordered state to ordered state.” I\ncould not understand this sentence when I saw Fig. 4a, b.\nWe thank the reviewer for the comment and apologize for the unclear discription.\nWe have revised the text as follows: “The R-T/S-W motif locates right before TMH1\n(W445-Y469 in Fks1). The side chains of T444 and W445 face the active site on\nthe right in the apo state but turn left in the Fks1-EFU-Lip state, flipping by nearly\n180°. Conversely, the side chain of W445 faces leftward in the apo state but\nswitches to the right in the Fks1-EFU-Lip state.”\n9. P8L208. TMH1 =Transmembrane helix 1?\nThanks for the comment. We have revised the TMH1 to TM1 (Transmembrane\nHelix 1).\n10. P8L214. “R443L” In Fig. 4b, there is only R443A.\nWe thank the reviewer for the comment and apologize for the mistake. The mutant\nhas been corrected to R443A in the text, consistently with Figure 4d.\nMinor points:\n1. P2L54. naturally triterpene -> natural triterpene\nThanks for the comment. We have revised it.\n2. P3L69 C-terminal of FKS1 or FKS2 -> C-terminus ? FKS1 and FKS2 are written\nin italics and seem to be gene names, but “C-terminus/C-terminal” is only\napplicable for polysaccharides (proteins).\nThanks for the comment. We have revised this sentence to “Endogenous Fks1 and\nFks2 were both purified from yeast cells with triple FLAG epitope tagged on their\nC-terminuses.”\n3. P3L73. .. and the lauryl -> and lauryl\nThanks for the comment. We have revised it.\n4. P3L73-74. .. (LMNG) and CHS were used ..\nThanks for the comment. We have revised it.\n5. P3L79. .. activity in (under?) the same condition\nThanks for the comment. We have revised it.\n6. P3L80. .. may function using ..\nThanks for the comment. We have revised it.\n7. P8L211. “ion bonds” salt bridges?\nThanks for the comment. The phrase has been revised.\n8. P8L221. .. but relatively lower ..\nThanks for the comment. The phrase has been revised.\n9. P9L238. .. may indicate the ..\nThanks for the comment. The phrase has been revised.\n10. P8L249-250. .. that specifically inhibits ..\nThanks for the comment. The phrase has been revised.\n11. P8L255. basing on -> based on?\nThanks for the comment. The phrase has been revised.\nReviewer #2 (Remarks to the Author):\nA hallmark of successful clinical management for invasive fungal disease is the\navailability of effective antifungal therapy, and glucan synthase inhibitors\nrepresent one of the most important drug classes. The echinocandins, approved\nfor clinical use nearly 25 years ago, remain first-line therapy for a broad range of\ncandidiasis and candidemia. Although they are limited to intravenous\nadministration, they have recently been joined by the first-generation orally\nbioavailable enfumafungin derivatives, which also inhibit glucan synthase. Genetic\nstudies of resistance suggest that the enfumafungin drug binds to the glucan\nsynthase complex at a distinct yet partially overlapping site compared with the\nechinocandins.\nExtensive knowledge has accumulated regarding the genetics, resistance\nmechanisms, and biochemical and kinetic properties of glucan synthase, encoded\nby the FKS genes. However, until recently, little was known about the enzyme’s\nstructural organization or the molecular basis of drug–target interactions\nunderlying inhibition—information critical for optimizing antifungal therapy.\nRecent cryo-EM studies from this group and others have begun to illuminate these\nstructural features, especially concerning resistance-associated amino acid\nsubstitutions. Yet, our understanding of how echinocandins and related\nenfumafungin drugs achieve target inhibition remains largely inferential, based\nprimarily on genetic analyses of resistance.\nThis new study by a leading glucan synthase structural biology group provides\nmuch-needed insight into the structural basis of drug–target interactions\nresponsible for enzyme inhibition. The authors resolved five cryo-EM structures\nof Fks1 and Fks2 in the apo state, in complex with the triterpenoid enfumafungin\ndrug, as well as in an open state induced by echinocandin drug caspofungin.\nStructural analysis along with strategic mutagenesis revealed the putative\nenfumafungin binding site, and the likely mechanism for clinically important drug-\nresistant mutations. Consistent with inferences from numerous genetic studies in\nthe literature and some new mutant analysis, it is proposed that the enfumafungin\nattaches to transmembrane helix TM5 of the glucan synthase, reorganizes its\nnearby lipid environment, and stabilizes the enzyme in a unique basal state with\nintact active site. New insights about the location of the glucan channel, which\nappears to lie between TMDs 1 and 2 are exciting.\nThe elucidation of a structure from cryoEM studies with the enfumafungin bound\nis a key advancement of this work. Furthermore, the observation of an elongated\nextra density around the TM5 of Fks1 and Fks2 in the transmembrane region,\nwhich aligned well with the EFU molecule, was a major finding.\nOverall, this is an important contribution that strongly advances the field, and\nsupports more advanced computationally assisted drug design to develop next\ngeneration drugs that\noptimize enzyme inhibition across a range of high-threat fungi and have the\npotential to overcome FKS hot-spot resistance known to cause refractory drug\nresponses in patients.\nWe thank the reviewer for the positive comments of our work.\nRevision Considerations\nThe manuscript would benefit from some minor English editing:\nLine 194: Hot spot 2 effect the inhibitoty function. Please correct to: Hot Spot\n2 affect inhibitory function\nThanks for the comment. The sentence has been revised accordingly.\nLine 203: Should be: Therefore, it’s still unclear whether the FKS catalyze following\na similar …\nThanks for the comment. The sentence has been revised accordingly.\nLine 79. ‘It indicates Fks2 may functions using a different mechanism from Fks1.’\nIt is well-known that FKS1 and FKS2 have different regulation and potential\nfuncton(e.g., Healey et al. 2020; Katiyar et al. 2012; Garcia-Effron et al. 2009;\nGonzalez-Jimenez et al, 2025; Suwunnakorn et al. 2018)\nWe thank the reviewer for the constructive comment. We have added related\nbackgrounds in the second paragraph and cited the references.\nMoreover, we realized the statement was inappropriate and deleted it in the\nrevised manuscript. The statement that “Fks2 may function using a different\nmechanism from Fks1” was proposed basing on our initial failture to detect Rho1-\ndependent activity of Fks2, but we recognized that the failture was probably\ncaused by the use of Rho1-Q68H mutant, and detected the activity of Fks2 with\nwild-type Rho1 during the revision. The interaction between Fks2 and Rho1 was\nalso proved by the Crosslinking and EM analysis. Details can be found in the the\nresponse to reviewer1 (Response Fig. 1) and revised manuscript. We sincerely\napologize for the mistake.\nLine 83. It suggests the regulatory subunit of Fks2 is probably Rho3. This is a\nhelpful and insightful observation. Do we understand how Rho activates from a\nstructural perspective?\nThanks for the comment.\nPrevious studies have shown that Rho1 is essential for activity of the 1,3-β-glucan\nsynthase both in vivo and in vitro (ref. 9-13). Cryo-EM structure of FKS1 in\ncomplex with Rho1 has been reported earlier this year (ref. 14). In the structure,\nRho1 is positioned within a pocket between the glycosyltransferase domain of\nFKS1 (GT domain) and the transmembrane helix spanning TM7-15 (TMD2).\nComparison between apo and Rho1 bound states of Fks1 reveals extensive\nconformational changes. Authors suggested Rho1 ’ s GTP/GDP cycling may\npromote a dynamic transition between the resting and active states of FKS1,\ninduce the protein as a molecular pump to push the growing glucan chain into\nFKS1’s transmembrane channel.\nWe have incorporated all above mechanisms in the background of the manuscript.\nIn addition, we detected the activity of Fks2 with wild-type Rho1 during the\nrevision, and changed our speculation of regularotgy subunit of Fks2 back to Rho1.\nDetails can be found in the the response to reviewer1 (Response Fig. 1) and revised\nmanuscript. We sincerely apologize for the revision.\nIt was reassuring to see that the enfumafungin binding site was largely overlapped\nwith the echinocandin resistance “hot spot” regions, which aligns very well with\npublished genetic studies, as indicated.\nWe thank the reviewer for the positive comment.\nLines 191-195. The potential role of a dual sterols in interacting with\nenfumafungin and drug resistant mutants is intriguing. However, azoles which\nblock sterol biosynthesis and levels in the membrane are not known to have\nantagonistic nor synergistic effects on echinocandin nor enfumafungin action. The\nauthors may want to speculate about this known observation.\nWe thank the reviewer for the valuable suggestion, and totally agree that the\ncombined use of azoles and echinocandins or enfumafungin is indeed worth\ndiscussing.\nWe performed literature search on publications of combined use of antifungal\ndrugs. As the reviewer pointed out, most literatures support that azole-mediated\ninhibition of sterol biosynthesis has neither synergistic nor antagonistic\ninteractions with echinocandins or ibrexafungerp (the semi-synthetic derivative of\nenfumafungin) (ref. 49-51). In meanwhile, some literatures showed that the\ncombination of ibrexafungerp plus azoles was synergistic for the tested\nAspergillus fumigatus strains in vitro and in animal models (ref.52-53). We\npreviously showed sterols help to stabilize the overall Fks1 structure as purified\nFks1 in buffer without CHS forms aggregates predominantly (ref. 23), and futher\nsuggested the potential role of sterols in interacting with enfumafungin in this\nmanuscript. Whether and how the combined use of azole and ibrexafungerp has\nsynergistic effect would be a complex matter. We speculate that although sterols\nare involved in the structural stabilization of the Fks1/2 and triterpenes, whether\nthe azole-mediated inhibition of sterol biosynthesis affect the function of FKS and\ntriterpene drugs still awaits future research. These discussions have been added\nin the Discussion part of the revised manuscript.\nLine 255. Did the authors look to see if caspofungin could displace or compete\nin enfumafungin bound density observed from the enfumafungin bound? This\nwould get partially at the point of an overlapping binding site.\nThanks for the valuable suggestion.\nDuring the revision, we have tried to address this question by incubating purified\nFks1/2-EFU complex with caspofungin, and performing cryo-EM analysis.\nUnfortunately, we failed to get any structure in high resolution after try different\nincubation time and caspofungin concentrations. It seems direct addition of\ncaspofungin perturbs the stabilization of Fks1/2-EFU complex for structure\ndetermination. The experiments need more extensive parameter optimization and\nwe apologize for this. We hope to answer this question in future.\nThe general Discussion is sparse and should be expanded to discuss opportunities\nand alignment with genetic and biochemical studies. It would benefit from a\ndescription of study limitations. It is important note that in general structural\nresolution of 2.5–3.0 Å allows side chains to be visualized, but hydrogen positions\nare uncertain. For some structures at or above 3.5 Å, only backbone and bulky\nside chains are clear, while any atomic interactions must be inferred.\nWe thank the reviewer for the constructive suggestions regarding expansion of\nthe Discussion. In the revised manuscript, we have added several passages to\naddress this point:\n1. Resolution-dependent interpretability and limitations.\nWe now explicitly state that, side-chain placement is well supported by density at\ncurrent resolutions of our maps, whereas hydrogen positions are not directly\nobserved. Therefore, specific atomic interactions mentioned above are mainly\ninferred. This clarification has been incorporated into the Discussion to avoid\nover-interpretations.\n2. Potential role of sterols for FKS and combined use of antifungal drugs.\nAs mentioned in above response, we summarized the potential role of sterols for\nFKS and drugs, and discussed the combined use of antifungal drugs.\n3. Structutal insights into the Fks2.\nIn S. cerevisiae, Candida albicans and many other pathogenic fungi, FKS1 or FKS2\nalone is not essential, but their simultaneous knockout leads to lethal.\nFurthermore, mutations in both Fks1 and Fks2 have been reported to be\nassociated with echinocandin and triterpene drug resistance. These suggest the\ndrugs work by inhibiting both Fks1 and Fks2 in the cells. This study for the first\ntime provided the structural insights into Fks2. The conserved structures between\nFks1 and Fks2 indicated the echinocandin can inhibit both enzymes in a similar\nmechanism, and explained the overlap of echinocandin resistant mutations in\nFks1 and Fks2. The structures of enfumafungin bound Fks1 and Fks2 also support\nthe two enzymes share the same inhibition and drug resistant mechanisms of\ntriterpenes.\nAlso, the structural studies were conducted on the Saccharomyces cerevisiae\nglucan synthase, which is sensitive to enfumafungin but weakly sensitive to\nechinocandins. The authors did attempt to affirm the structural binding motifs\nwith relevant mutant enzymes from clinically significant pathogenic Candida\nspecies, but it would be valuable to understand potentials gaps in the\nanalysis. This particularly important in terms of interactions with sterol, bound\nlipids and impact on enzyme inhibition and resistance. While alpha-fold may be\nof limited value here, what about other computational modeling approaches? Were\nany energy minimizations run with bound drug for wild-type and mutant enzymes?\nThanks for the valuable suggestion.\nFirstly,we predicted structure of C. albican Fks1 in complex with EFU. The EFU\nmolecule was also wrongly placed in the active site as in the S. cerevisiae Fks1,\nindicating similiar prediction ability of AlphaFold3 for Fks1 homologues\n(Supplementary Fig. 14).\nSecondly, we performed extensive molecular dynamics (MD) simulations of the\nwild type and mutant Fks1 in complex with enfumafungin embedded in a lipid\nbilayer (Supplementary Fig. 8). Four EFU resistant mutations E635A, F639A, F639S,\nand W695L were selected for the MD simulations. RMSD values of enfumafungin\ncalculated relative to the starting structure were used to explore the stability in\nthe active sites of the wild type or mutant Fks1. The results revealed that the EFU\nis more dynamic in the mutants compared to WT, while the backbones of all\nresidues in both WT and mutant Fks1 are stable. The differences indicate that\nthese mutant sites are crucial for EFU binding.\nReviewer #3 (Remarks to the Author):\nIn this work, You and co-workers provide cryoEM structures of β-1,3-glucan\nsynthases Fks1 and Fks2 in the apo state, the triterpenoid drug enfumafungin-\nbound state, and an open state induced by echinocandin incubation. It is worth\nmentioning that structures of Fsk1 were previously reported (Li et al. Cryo-EM\nstructure of the β-1,3-glucan synthase FKS1-Rho1 complex. Nat Commun.\n16:2054 (2025); Zhao et al. Structure of a fungal 1,3-β-glucan synthase. Sci Adv.\n9:eadh7820 (2023); Hu et al. Structural and mechanistic insights into fungal β-\n1,3-glucan synthase FKS1. Nature 616(7955):190-198 (2023). In combination\nwith single-point mutagenesis, activity measurements, growth complementation\nand antifungal susceptibility assays, the authors provide new and important\ninsights on the molecular basis of catalytic cycle, activation by Rho1, and inhibition\nby triterpenoid and echinocandins of β-1,3-glucan synthases. The article is well\nwritten and the interpretation of the experimental data support the proposed\nmodel. Therefore, I support publication of the manuscript in Nature\nCommunications.\nWe thank the reviewer for the positive comments.\nSome suggestions for improvement are listed below:\n- page 2, line 38: I would suggest to replacing ““membrane bound enzyme” by\n“integral membrane enzyme”.\nThanks for the comment. The phrase has been revised.\n- page 2, line 39: “catalyzing the synthesis of β-1,3-glucan”. Please, incorporate\nthe reaction scheme as Figure 1a.\nWe thank the reviewer for the helpful suggestion. We have prepared the reaction\nscheme and incorporated it as Figure 1a.\n- page 2, line 40: “Fungal β-1,3-glucan synthase is primarily encoded by FKS1 or\nFKS27, the activity of which was regulated by Rho1”. Please, elaborate the main\nsimilarities/differences and how Rho1 regulates the enzymes.\nThanks for the suggestion. We have incorporated the main similarities and\ndifferences between FKS1 and FKS2, and how Rho1 regulates these enzymes in\nparagraph 2 in the revised manuscript.\n- page 2, line 46: “in presence of a 4-glucose glucan have been determined”.\nPlease, revise and incorporate the precise identity/nomenclature.\nThanks for the comment. The “4-glucose glucan” was used following the original\nreference (ref.24). Because the structure was determined using sample incubated\nwith UDP-Glc, authors proposed it to be a product-bound state of the enzyme.\nThe glucan was modeled basing on the density, which fits well with 4-glucoses.\nTo be more clear, we revised the “in presence of a 4-glucose glucan” to “in\ncomplex with glucan product”.\n- page 2, line 47: Please, incorporate FKS1 and FKS2 belongs to CAZy family GT48.\nIt would be interesting the authors could elaborate - and also include a sequence-\nbased structure alignment, highlighting important residues involved in\ncatalysis/beta-glucan transport and protein-protein/protein-inhibitors\ninteractions - on the homology of FSK1/FSK2 with other enzymes adopting a\nconserved GT-A type glycosyltransferase domain and a transmembrane glycan-\ntransporting channel - e.g. cellulose synthase, chitin synthase and hyaluronan\nsynthase.\nThanks for the suggestion.\nFirstly, we have incorporated the information that FKS1 and FKS2 belong to the\nCAZy family GT48 in the second paragraph.\nSecondly, we performed structural alignment between Fks and other membrane-\nbound glycosyltransferases, including cellulose synthase, chitin synthase, and\nhyaluronan synthase, and incorporated the analysis in the section of “Intact active\nsite and glucan transport channel in the structures of Fks1/2 in complex with EFU”\nas Supplemental Fig. 10. These enzymes share a generally conserved fold in active\nsite and glucan transport channel, while structure-based sequence alignment\nshowed low similarity. There are only a few conserved residues identified, such as\nthe magnesium coordinating residue D1102 and the phosphate coordinating\nresidue R1261. This is probably due to the different substrates and functions of\nthe enzymes. The analysis have been incorporated into the maintext.\n- page 3, line 76: “Functional characterization with in vitro UDP-Glo\nglycosyltransferase assay”. Please, confirm the use of UDP-glucose as sugar donor\nin the Methods section.\nThanks for the comment. We have revised the Methods by adding the UDP-glucose\nin the reaction system.\n- page 4, line 91: “The overall structures of Fks1-apo and Fks2-apo are similar to\neach other with a root-mean-square-deviation (RMSD) of ~1.5 Å over 1449\nresidues”. Please, state UniProt codes and total number of residues for both\nenzymes.\nThanks for the suggestion. We have added the UniProt codes and total number of\nresidues for both enzymes.\n- page 4, line 100: “… a glycosyltransferase (GT) domain inserted between TM6\nand TM7…” A good opportunity to introduce a topology model on Fks1/2 as\nFigure 1f.\nThanks for the suggestion. We have prepared a topology model and added it as\nFig. 1f for better illustration of Fks1/2.\n- page 5, line 128: “EFU was found stacking against the TM5 and embedding in\nthe exoplasmic leaflet of the lipid bilayer”. Please, could the authors state how far\nis the EFU binding site from the beta-glucan-transporting channel - entrance/exit\n- of Fks1 and Rho1? Please, incorporate an additional panel in Figure 2 and/or\nFigure 4, including the β-1,3-glucan synthase in surface representation - e.g. use\nPDB 7YUY and 8WLA as references - different orientations - cytosolic/extracellular\nentrances - to better show the channel, and secondary structures involved.\nThanks for the suggestion.\nWe modeled the glucan and Rho1 into the structure of Fks1-EFU by performing\nstructural superposition of EFU bound Fks1, glucan bound Fks1 (PDB 7YUY) and\nRho1 bound Fks1 (PDB 7YUY), Then, we measured the distances between EFU\nmolecule and the active site or putative glucan transporting channel, prepared a\nnew figure as Figure 2g. Description of this new panel was inserted in the maintext\nas follows:“The EFU molecule locates ~30Å from the active site and ~25 Å from\nthe putative β-1,3-glucan–transporting channel of Fks1 (Fig. 2g).”\nPlease, also check:\n- page 1, line 25: please replace “enviroment” by “environment”.\nWe thank the reviewer and apologize for the mistake. The word has been revised.\n- page 2, line 44: please homogenize FKS1 and FKS2 or Fks1 and Fks2, when\nappropriate.\nThanks for the comment. We have homogenized the gene and protein names\nthroughout the manuscript with gene names as FKS1/2 (italic, uppercase) and\nprotein names as Fks1/2.\n- page 7, line 194: please replace “inhibitoty” by “inhibitory”.\nThanks for the comment. The word has been revised."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised manuscript, the authors have added new data, such as the regulation of Fks2 by Rho1 and the detection of the open state, which lead to more robust and convincing conclusions. The points I raised have been fully addressed.", "reviewer_2": "I want to commend the authors for their rigor in addressing all of the concerns and suggestions raised by the reviewers. In particular, the dynamic reassessment of the Rho3-FKS2 activation complex was most welcome. Overall, the manuscript has been significantly improved and will make a welcome contribution to the field"}, "author_response": ""}]

Decision letter