Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

Domain: physics
Published: 2025-11-26
Editorial outcome: major_revision

Abstract
Acriticalstepininfectionsistheattachmentofmicroorganismstohostcells
usinglectinsthatbindglycans,makinglectinspromisingantimicrobialtargets.
Uponbindingmannosylatedglycans,FimH,anadhesininE.coli,undergoesan
allosterictransitionfromaninactivetoanactiveconformationthatcanactasa
catch-bond.DistinctmonoclonalantibodiesthatalterFimHglycanbindingare
available,butthemechanismsofactionremainunclear.Here,weusecryo-
electronmicroscopy,massspectrometry,adhesionassays,andmolecular
dynamicssimulationstodeterminethestructure-functionrelationships
underlyingantibody-FimHbinding.Ourstudydemonstratesfourmechanisms
ofaction:ligandmimicrybyanN-linked,high-mannoseglycan;stabilizationof
theligandpocketintheinactivestate;conformationaltrappingoftheactive
andinactivestates;andlockingoftheligandpocketthroughlong-range
allostericeffects.Thesestructuresrevealmultiplemechanismsofantibody
responsestoanallostericproteinandprovideblueprintsforantimicrobials	hattargetadhesins.

Introduction
Microbesadheretosurfacesasthefirststepintheinfectionofahost
and in the formation of biofilms on both biological and abiotic
surfaces1. Adhesion also facilitates the mutualistic relationships
foundinthegastrointestinaltractsofvariousspecies2.Manyviruses,
bacteria,fungi,and parasites use lectin-based cellattachment pro-
teins during infection or surface adherence, recognizing specific
moietiesonlinearorbranchedoligosaccharidechainsonthetarget
surfaces3.
FimH,fromEscherichiacoli,isthebestcharacterizedmemberof
the largest family of structurally diverse fimbrial adhesins found in
Gram-negativebacteria,whichareassembledviathechaperone-usher
pathway4 (Supplementary Fig. 1A, B). In E. coli, FimH acts as a key
virulence factor in urinary tract infections and inflammatory bowel
disease.FimHislocatedatthetipoftherod-shapedtype1fimbriae
(pili) and contains two immunoglobulin-like domains: the lectin
domain,whichbindsα-D-linkedmannosesofN-linkedglycans;andthe
pilindomain,whichconnectsthelectindomaintoadditionalsubunits
onthefimbria5–9.
FimH also serves as a model allosteric protein as it undergoes
dramatic conformational rearrangements that decrease ligand
dissociation7,10.Intheabsenceofligandandtensilemechanicalforce,
theFimHlectindomainadoptsacompressedandinactiveconforma-
tion stabilized by a fixed interface between the lectin and pilin
domains,withtheligand-bindingpocketrelaxedandopen10(Supple-
mentary Fig. 1C). FimH can adopt an intermediate conformation,
wherealigandoccupiesthebindingsitebutmechanicalforceisnot
applied.Inthisconformation,themannose-bindingloops,N-terminus,
andclamploopencircletheligand,butthelectin-pilindomaininter-
faceremainsfixed11(SupplementaryFig.1D).Ligandbindingwithout
mechanicalforcealterstheequilibriumconstantoflectin-pilindomain
separationinFimH11–13.ThischangeincreasestheproportionofFimH
that adopts an elongated active conformation where the pilin and
lectin domains are separated, although the domain-separated con-
formation of full length, ligand bound FimH is unstable and less
populatedintheabsenceofforce(SupplementaryFig.1E).Allosteric
modulation by tensile mechanical force causes lectin-pilin domain
separation,pullingthepilindomainawayfromthelectindomainand
stabilizingconformationalrearrangementsandligandbindinginthe
lectindomain.Theresultisaforce-enhancedcatch-bondinteraction
betweentheligandandthelectindomain7,10,14,15.
Becauseofthecriticalroleadhesinsplayasvirulencefactorsin
infectionandbiofilmformation,adhesinsareattractiveantimicrobial
targets. Several strategies for inhibition of adhesins have been
explored. For example, researchers have created compounds con-
taining mannose and other glycan motifs to competitively inhibit
surfaceglycanbinding,astrategysimilartomammaliansecretionof
mucins and oligosaccharides to prevent pathogen binding16–23.
ResearchershavealsousedFimHandotheradhesinsasvaccineanti-
gentstoelicitneutralizingantibodies24–26.Follow-upstudies,however,
demonstrate that the polyclonal antibody response is complex and
varies depending on the specific form of FimH used for
vaccination11,24,27,28.
Our previousstudiesidentifiedmonoclonalantibodies (mAbs)
thatimpacttheallostericpropertiesandneutralizeFimHviadistinct
mechanisms29. We previously classified the monoclonal antibodies
intofourcategories:orthosteric,parasteric,dynasteric,andactivat-
ing. Orthosteric antibodies, like mAb475, directly compete for the
ligand binding pocket and behave like classical competitive
inhibitors27.mAb475actssimilarlytoFimHligands;thecoreepitope
includesmannosebindingresiduesonFimHandtheantibodytrig-
gers the closing of the ligand binding pocket and shifts the con-
formational equilibrium (Supplementary Fig. 1F). In contrast,
parasteric antibodies (mAb926) exhibit non-competitive behavior
and the core epitope includes a few mannose-binding residues as
wellasnearbyresidues27(SupplementaryFig.1F).mAb926stabilizes
the inactive conformation of FimH and the antibody can uniquely
elute bacteria already attached to a mannose-coated surface.
Dynastericantibodies,(mAb824),arresttheconformationaldynam-
icsofFimH;theantibodiesbindbothactiveandinactiveconforma-
tionsofFimH,butpreventthetransitionbetweenconformations28.
mAb824bindingoftheinactiveconformationpreventsFimHfrom
productivelyengagingwithglycans,evenundershearstress28.The
core epitope for mAb824 is distal to the ligand-binding pocket
(SupplementaryFig. 1F,G).Activating antibodies, like mAb21, only
bind the active conformation of FimH24,27,28. The core epitope for
mAb21 includes residues in the interdomain region of the lectin
domain(SupplementaryFig.1F).Whileourpreviousstudiesprovided
insights into the binding epitopes and large-scale conformational
rearrangements that occur upon antibody binding, we lack the
understanding of how each antibody exerts its effect at the mole-
cular level, information which is required for the development of
therapiesagainstFimHandothercellattachmentproteinsofhuman
pathogens.
Here,weinvestigatethemechanismsofactionforfourfunction-
ally distinctantibodies. The orthosteric mAb475 leverages an unex-
pected N-linked, high-mannose glycan on a hypervariable loop to
mimic the native, glycosylated host cell receptors to competitively
block adhesion. The parasteric mAb926 occupies the open ligand-
binding pocket yet retains partial flexibility in the antibody hyper-
variable loops which generates non-competitive inhibition. The
dynasteric mAb824 directly engages allosteric residues on FimH to
block conformational transitions. The activating mAb21 acts as a
molecularwedgebetweenthelectinandpilindomains,preventingthe
lectin-pilininterfacefromreforming.EachFimH-antibodyinteraction
providesmechanisticinsightswithimplicationsforthedevelopment
ofanti-adhesintherapiesandforhumanresponsestothecell-binding
proteinsofpathogens.

Results
Cryo-EMstructuresrevealbindingmodesandFimH
conformations
We used single-particlecryo-EM to determine structures of fimbrial
tips in complex with antibody fragments (Fabs) (Supplementary
Figs.2–8,SupplementaryTables1–3).Wecombinedfullfimbrialtips
(FimH/G/F with chaperone FimC) with multiple Fabs without over-
lappingepitopes.Thissamplepreparationstrategyincreasedthesize
of our target while allowing us to unambiguously identifyeach Fab
boundtoFimH(Fig.1A–E).Wecouldnotresolvecomponentsofthetip
beyondFimG,likelyduetoflexibility(SupplementaryFig.3B–F).Both
Fab926andFab475bindthemannosepocketofFimHbutindistinct
orientations relative to FimH (Fig. 1A, C, Supplementary Fig. 9A).
Fab824bindstothesideofthelectindomain(Fig.1A–E,Supplemen-
taryFig.9A).Fab21interactswiththelectindomainatthelectin-pilin
domaininterface(Fig.1D,E,SupplementaryFig.9A).Thisagreeswith
previous mutational analyses that identified the functional epitope
residuesrecognizedbyeachofthefourantibodies24,27,28(Supplemen-
taryTable4,SupplementaryFig.1F,G).
AllFimHconformationsobservedareconsistentwithpriorcrystal
structures7,10,12,24,27,28 (Fig. 1F–J). For both the FimH-Fab475/824 and
FimH-Fab824/926complexes,thelectinandpilindomainsarefixedin
thedomain-interactingconformationata94°angle(Fig.1F,H).The
clamp loop, which forms the external face of the ligand-binding
pocket, remains open in the FimH-Fab824/926 complex, and FimH
adoptstheinactiveconformation(Fig.1F,SupplementaryFig.7C).In
contrast,thisloopisclosedinthepresenceofFab475,andtheFimH-
Fab475/824complexadoptstheintermediateconformation(Fig.1H,I,
Supplementary Fig.7C). The lectin and pilin domains are separated
andflexiblerelativetoeachotherinthethreecomplexescontaining
Fab21:FimH-Fab21/475;FimH-Fab21/475/824;andFimH-Fab21/824/α-
methylmannose(Fig.1G,H,SupplementaryFig.9B,Supplementary
Movie1).AllthreecomplexeswithFab21areintheactiveconforma-
tion,whileFab824canbindallthreeconformations.

AglycanonmAb475competitivelyinhibitsligandbinding
Tooursurprise,thecryo-EMdatarevealedaglycanlinkedtoAsn90on
theFab475hypervariableloop3(complementaritydeterminingregion
3;CDR3)intheglycan-bindingpocketofFimH(Fig.2A).Thevolume
supportstheplacementoffivesugarsfromanN-linkedglycaninthe
structure,withthetwosugarsproximaltoAsn90likelycontaining
N-acetylgroupsandtheFimHpocketcontainingmannose.Boththe
Asn90glycanonFab475andtheresiduesintheFimHligandpocket
adoptpositionsverysimilartoFimHlectindomainstructuresbound
to oligomannose-3, recapitulating glycan and mannose binding6,8
(Fig.2A,B,SupplementaryFig.9C).Thisresultrationalizesthecom-
petitive inhibition seen between mAb475 and mannose, and how
mAb475 shifts the conformational equilibrium to the active con-
formation,similartosolublemannose.
ToidentifyandquantifytheglycosylationsontheFabsofeach
antibody,wedigestedtheFabs with either trypsinor chymotrypsin
andanalyzedtheresultingintactglycopeptidesusingmassspectro-
metryonFabsfromeachantibody(Fig.2C,SupplementaryFigs.10,11).
Only Fab475 exhibited N-glycosylation on its light chain, with two
glycosylated sites present in the light chain CDRs (Table 1, Supple-
mentaryData1–3).Thefirstsite,Asn90inCDR3,directlyinteractswith
FimHasseeninthecryo-EMdata.Massspectrometryidentifiedonly
threeglycancompositionsattachedtotheasparagine(Fig.2C,Sup-
plementaryData1).Twoofthesearehigh-mannoseglycans,andthe
third is likely a hybrid sialylated glycan. The second site, Asn28 in
CDR1,doesnotinteractwithFimH.Asn28showedadiverseportfolio
of N-linked glycans;however,over 95%of the glycan intensity con-
sistedofsialylatedglycans(SupplementaryFig.10A,Supplementary
Data2).Inspectionofthetwo-dimensionalclassaveragesandthecryo-
EMvolumesurroundingAsn28showedadditionaldisorderedvolume
in agreement with the mass spectrometry data (Supplementary
Figs.3Gand10B).Investigationofdeamidatedasparaginesgenerated
byPNGaseFtreatmentcorroboratestheidentifiedglycansandthelack
ofN-glycosylationontheotherFabs(SupplementaryData3).Further
confirmationofglycosylationonFab475andtheabsenceofglycosy-
lationontheotherFabswasobtainedthroughnativemassspectro-
metry analysis. Intact analysis of Fab475 revealed highly
heterogeneous spectra that eluded simple deconvolution, which is
characteristic of glycosylated proteins (Supplementary Fig. 10C). In
contrast, Fab926, Fab824, and Fab21 revealed less complex and
interpretable spectra with no indication of glycosylation (Supple-
mentaryFig.10D–F).
Whiletheglycanonlyaccountsforhalfoftheburiedsurfacearea,
it dominates the mAb475-FimH interaction. In total, Fab475 buries
712Å2 of the FimH pocket (Supplementary Fig. 9A, Supplementary
Table4).TheglycanonFab475buries372Å2ofsurfacearea,whichis
nearlyidenticaltotheburiedareaofaboundglycanalone(PDBID
2VCO, 379Å2)6. Dissociation constants for oligomannose-3 and
oligomannose-5 are 10–20nM, suggesting that the boost in affinity
observed forFab475(4nM)arises from additionalinteractionswith
theantibody6,27.Theremainderoftheinteractionsoccurbetweenthe
hypervariable loops in the heavy chain of Fab475 and FimH. These
contactsareprimarilymadewiththeoutersideandtopoftheman-
nose binding loop 136–140 through a series of polar interactions
(SupplementaryFig.9D).Usingbothitscovalentlyattachedglycanand
heavy chain residues, Fab475 grabs the mannose binding loops of
FimHintheactiveconformationlikepincers(Fig.2D,E).
WeassessedthecontributionsoftheglycansinmAb475binding
and inhibition of FimH by deglycosylating mAb475 and performing
bindingexperiments(Fig.2F,SupplementaryFig.12A).Unlikethelight
chains of the other three antibodies, the light chain of mAb475
migratedat~30kDaonSDS-PAGE,5kDaheavierthanthepredicted
25kDa (Supplementary Figs. 12A, 13). Treatment of mAb475 with
endoglycosidaseS(EndoS),whichcleavessialylatedN-linkedglycans
likethosefoundonAsn28,shiftedthelightchaintoalowermolecular
weight. As expected, removal of this glycan did not affect mAb475
binding to FimH (Supplementary Figs. 12A and 13). Treatment of
mAb475 with endoglycosidase H (Endo H), which cleaves high-
mannoseandsomehybridglycanslikethosefoundonAsn90,shif-
tedaportionofthelightchaintoalowermolecularweight,indicating
partial removal of the targeted glycan. This partial deglycosylation
decreased mAb475 binding to FimH (Supplementary Fig. 12A, Sup-
plementaryFig.13).TreatmentwithbothEndoSandEndoHshifted
themajorityofthemAb475lightchainclosertothepredictedsizeof
25kDa (Supplementary Figs. 12A and 13) and decreased mAb475’s
abilitytobindFimH(Fig.2F,SupplementaryFig.12A).Thepatternand
multiple species in the mAb475 digestions with EndoS, EndoH, and
EndoS/EndoHshowsthatwemaynotachievefulldeglycosylationat
Asn90(SupplementaryFigs.12A,13).Combinedwiththenon-linearity
of the ELISA assay, the assay limitations likely lead to an under-
estimation of the magnitude of glycan contribution to antibody
binding(SupplementaryFig. 14). Alongwith the mass spectrometry
andcryo-EMresults,thebiochemicalresultsconfirmthatthecomplex
glycansprimarilyfoundonAsn28donotcontributetoFimHbinding
whilethehigh-mannoseandhybridglycansonAsn90drivebinding
andinhibitionbymAb475.

mAb475-likeantibodiesarenaturallyelicited
TheFimH-Fab475interactionpresentsanantigen-antibodyinterface
where a hypervariable-loop glycan is specifically recognized by the
targetlectin,allowingtheantibodytodirectlyblocktheligand-binding
site. However, mAb475 is not unique; we previously identified six
additionalantibodieswithcompetitive,orthostericbindingbehavior
similar to mAb47524. Sequence alignments of these antibodies and
closelyrelatedsequencesfrompublicdatabasesshowthattheanti-
bodies from our study contain a glycosylation motif (NxS/T, x=all
aminoacidsexceptproline)atthealignedequivalentresidueofAsn90
(Supplementary Fig. 12B).Inaddition, the sequences segregate into
twogroupswiththesecondglycosylationsiteatAsn28appearingin
one group but not in the second. Together, this suggests that the
mAb475-likeantibodiesinourstudyoriginatedfromtwoindependent
germlinelineagesandfurtheraffinitymaturationproducedmultiple
glycosylatedantibodies.
WenextestimatedtheapproximatepercentageofmAb475-like
antibodies in the polyclonal antibody pool of mouse antiserum
obtainedbyimmunizationwiththeFimHlectindomain.Weselectively
depleted antibodies from aliquots of antiserum using bacteria
expressing:a)fimbriawithoutFimH;b)fimbriawiththeFimH-FocH,a
variantthatprimarilyadoptstheactiveconformation;andc)fimbria
withtheFimH-FocHF1Lmutant,wherethephenylalanineatposition1is
replacedwithleucine,eliminatingtheabilityofFimHtobindmannose
andglycans.Noneofthemonoclonalantibodiesrecognizedfimbriae
lackingFimH.AllfourantibodiesrecognizedFimH-FocH,butmAb475-
likeantibodiesdonotrecognizeFimH-FocHF1L(Fig.2G,Supplementary
Fig.12C).
After adsorption, we probed for FimH binding within the
remaining pool of antibodies in each antiserum sample (Fig. 2H,
SupplementaryFig.12D).AdsorptionwithbacterialackingFimHdid
not significantly decrease anti-FimH antibodies in the serum (Sup-
plementaryFig.12D).Inaddition,mannosehadnosignificanteffect
onunadsorbedantiserumorantiserumtreatedwithbacterialacking
FimH(SupplementaryFig.12D).Incontrast,thevastmajorityofthe
antibodypoolwasremovedbybacteriaexpressingFimH-FocHfim-
briae(SupplementaryFig.12D).Asmallbutmeasurableincreasein
the absorbance signal was observed in the presence of mannose,
indicating that some of the non-absorbed antibodies may be acti-
vating, like mAb21 (Supplementary Fig. 12D). When the antiserum
wasabsorbedwith bacteria expressing the FimH-FocHF1Lmutant,a
larger portion of the antibodies remained unabsorbed (Fig. 2H,
Supplementary Fig. 12D). Notably, the binding significantly
decreasedinthepresenceofmannose,indicatingthatthebulkofthe
non-adsorbed antibodies compete with mannose for binding to
FimH. This suggests the polyclonal antibody response contains a
minor but clearly detectable portion of orthosteric, mAb475-like
antibodies(Fig.2H).

mAb926stericallyblocksligandpocketclosure
In contrast to mAb475 binding within the closed ligand pocket,
mAb926bindstheopenligandpocketwithFimHintheinactivecon-
formation(Figs.1A,Fand2A,SupplementaryFig.7B,C).Thebinding
epitopeofFab926centersaroundthemannose-bindingpocketinthe
open conformation, with a total interaction area of 983Å2 (Supple-
mentaryFigs.9A,12A,SupplementaryTable4).BothchainsofFab926
formpolarinteractionsandhydrophobicpackinginteractionswiththe
innerandouterfacesofthemannosebindingloopsofFimH(Fig.2A,
SupplementaryFig.15A).ThisconfigurationpositionsFab926tobury
aminoacidsfromportionsofhypervariableloopsontheheavychainin
theopenligandbindingpocket.Additionalsectionsoftheparatope
stericallyblockclamploopclosurewithoutmakingspecificcontacts
withtheclamploop(SupplementaryFig.15B,C).Together,theheavy
and light chainsofFab926pinchthemannosebindingloopsofthe
ligandpocketwhiletheheavychainfillsandsplaysopenthebinding
pocket(Fig.2I).ThedifferencesinFimH’sconformationandbinding
epitopesbetweenmAb926versusmAb475explainwhytheantibodies
allostericallystabilizeinactiveandactivestatesofFimH,respectively.
mAb926 binding shows some similarities to both glycan and
mAb475 binding. As with Fab475, hypervariable loops on Fab926
contactthetopandtheoutersideofthemannosebindingloops.The
area buried in the ligand pocket by Fab926 overlaps with regions
previouslyshowntobeburiedbymannose,high-mannoseglycans,
andsmall-moleculemannosylcompounds6,19,30,31 (Supplementary
Fig.15D,E).HypervariableloopsonFab926recreateasubsetofthe
polar interactions generated by glycan ligands, including contacts
madebythemannosemoietydeepinthebindingpocketandothers
made by the N-acetylglucosamines with the upper portions of the
mannosebindingloops(SupplementaryFig.15E).Inaddition,several
hydrophobicresiduesonFab926contactahydrophobicpatchinthe
FimHpocket(SupplementaryFig.15F).Insum,thestructuredemon-
stratesthatfullyboundFab926stericallyoccludesbothnativeligand
binding and the closure of the binding pocket required for the
activestate.

ThemAb926-FimHinterfaceisflexible
Whilethecryo-EMstructuresshowhowmAb475andmAb926stabilize
differentFimHconformations,themodelsdonotexplainwhymAb926
inhibits FimH in a non-competitive manner. Previous work demon-
strated that mannose is about 30 times less effective at blocking
mAb926bindingascomparedtomAb475binding,eventhoughboth
antibodies’ epitopes include the ligand-binding site27 (Fig. 1A, B). In
addition,mAb926andmannosecanreciprocallydisplaceeachother
from FimH, which is not observed with mAb47527. These previous
resultssuggestmoreflexibilityattheFimH-Fab926interfacethanis
representedinoursinglestructure.
ToexploretheflexibilityofFab926binding,weusedmolecular
dynamics simulations. We performed simulations using either the
FimH-Fab926structureortheFimH-Fab475structuretodeterminethe
range of flexibility for each interaction (Supplementary Figs. 16, 17,
SupplementaryTable5).Wetrimmedcomputationalcostsbylimiting
our simulations to the lectin domain of FimH and the N-terminal
variabledomainβ-sandwichfromtheheavyandlightchainsoneach
Fab.PreviousstudieshaveshownthattruncationofFimHafterresidue
160hasminimaleffectsonthedynamicsofthelectindomaininthe
timeframeofthesimulations32,33.Thepersistenceofsidechaincon-
tactsbetweentheβ-sandwichesoftheheavychainandthelightchain
of the Fab interface over the course of the simulation indicate the
stability of the models (Supplementary Fig. 16F).Both mAb475 and
mAb926 interact with the clamp loop and stabilize the loop when
boundtotheFimHconformationobservedinthecryo-EMstructures
(Supplementary Table 6). mAb475 also showed a statistically sig-
nificantchangeinswingloopdynamics.Thisincreasefitswithprevious
workshowingthatligandbindingchangesthethermodynamiccycle
andalterstheequilibriumconstantsbetweenFimHconformationss7,11.
Over the course of a 50-ns simulation run at 330K, the FimH-
Fab475 interaction remained unperturbed, and the clamp loop
remainedclosed(SupplementaryFig.16A,B).Incontrast,thesimula-
tionwithFimH-Fab926showeddisruptionofinteractionsbetweenthe
FimHlectinbindingpocketandhypervariableloop1ofFab926(resi-
dues 26–33), which sits in the lectin domain pocket and above the
clamp loop (Supplementary Fig. 16C–F). The portions of this loop
outsideofthebindingpocket,aminoacids24–28,arenotwellresolved
in the cryo-EM structure, also suggesting flexibility (Supplementary
Fig.15B,C).CoincidentwiththemovementoftheFab926loop,the
clamploopofFimHmovestowardstheclosedpositionadoptedinthe
activeconformation,inthedirectionofFab926.Together,thesecon-
formationalchangescreatespaceformannoseinthebindingpocket
whilemAb926isstillboundtoFimH,highlightingtheflexibilitywithin
theFimH-mAb926bindinginterface(SupplementaryFig.16C).Mini-
mization of the model from the 50ns time point of the simulation
whileincluding α-methyl mannose shows the molecule forming the
appropriate hydrogen bonds and intermolecular distances (heavy
atom Fab926-Mannose distance: 2.9Å; heavy atom FimH-Mannose
distance: 2.7Å) within a pocket volume (263Å3) large enough to
accommodate the molecule volume(149Å3)with minimaldeviation
fromthesimulation(heavyatomRMSD:0.7Å,all-atomRMSD:0.8Å)
(Fig.2J,SupplementaryFig.16G,Table2).Oursimulationssuggestthat
mannosemaybindFimHwhileFab926isbound,andFab926maybind
when mannose is bound, with each destabilizing the binding of
theother.

FimHretainsinterdomainmobilitywhenboundtomAb21
ThestructurescontainingFab21supportpreviousworkshowingthat
mAb21bindsFimHintheactiveconformationatfunctionalepitope
residues located in the interdomain interface24,34 (Supplementary
Fig. 1E, Supplementary Table 4). The 721 Å2 epitope of Fab21 only
includesresiduesontheinterdomaininterfaceofthelectindomain
(Fig.3A,B,SupplementaryFig.9A,SupplementaryTable4).Theanti-
bodybindsinbetweentheFimHlectinandpilindomainsbutdoesnot
specificallyinteractwiththepilindomain(SupplementaryFig.9B).Our
simulationsdonotshowastatisticallysignificantchangeinloopflex-
ibilitywhenmAb21isbound;however,thiscouldbeduetothelimited
time scale of the simulations performed here35 (Supplementary
Table5).ThetwochainsofFab21pinchswingloopresidues23–29and
packtightly against the remainder of the swing loop and the inter-
domainloops(Fig.3A,B).Inthehigh-resolutionstructurescontaining
Fab21,Fab824,andeitherFab475orα-methylmannose,FimHisinthe
active conformationand Fab21’s position physically blocks the pilin
andlectindomainsfromreengagingandformingthefixedinterface
seen in the intermediate and inactive conformations (Supplemen-
taryFig.9F).
ThepilindomainremainsmobilewhenFab21isbound.Theclass
averagescontainingFab21andFab475showthepilindomaininmul-
tiple positions (Fig. 3A, Supplementary Movie 1, Supplementary
Figs. 3F and 9B). Reconstructing the high-resolution datasets con-
tainingFab21withoutmasksandfilteringthevolumesonlyresolvesthe
portion of the pilin domain proximal to the lectin domain, demon-
stratingthatitispresentbutblurredinthedatasets,asignatureof
mobiledomains(SupplementaryFig.9E).Fab21mayblockasubsetof
possible lectin-pilin orientations, but the lectin domain retains its
abilitytoflexandrotaterelativetothepilindomain.

mAb824trapsdynamicresiduesonFimH
Asshownpreviously,theFab824epitopespansthesurfaceoftheβ-
sheetdistaltotheligandbindingpocketoftheFimHlectindomain
(SupplementaryFig.1G,SupplementaryTable4).Thelightandheavy
chainsofFab824straddleresidues79-82and91,originallyidentifiedas
criticalinteractionsinamutationalepitopescreen28(Fig.3D,Supple-
mentary Fig. 1G). Additionally, the full contact surface of Fab824
bound to FimH contains residues thatwerepreviouslyidentified as
partoftheepitopeby13CH -NMR28(SupplementaryFig.1G).Theepi-
3
topedefinedbythecontactsurfaceinthecryo-EMstructureincludes
983Å2oftheFimHsurfaceintheinactiveconformation(Supplemen-
taryFig.9A,SupplementaryTable4).Thiscontactsurfaceislargerthan
theepitopeofFab824intheactiveconformation(854Å2,previously
defined by NMR; 839Å2, this study)28 (Supplementary Fig. 9A, Sup-
plementaryTable4).
Our simulations show that Fab824 stabilizes the clamploop of
FimHintheinactiveconformation(SupplementaryTable5).Incon-
trast, mAb824 does not show a statistically significant differencein
stability on the active clamp loop in the limited time scale of our
simulations, though the decrease in RMSD trends towards
stabilization. It is important to note that the clamp segment in the
active conformation is generally much more stable than in inactive
conformation33.Theseresultsagreewithourprevioushypothesesthat
mAb824stabilizestheconformationofFimHthatitbinds.
When bound solely to Fab824, FimH adopts the inactive con-
formation,whichsupportstheproposedmAb824mechanism.Inthe
cryo-EMdatasetscontainingcombinationsofFab21,Fab475,Fab824,
and α-methyl-mannose, we observe two-dimensional class averages
containingFimHandFab824(SupplementaryFig.3H).Weusedthe
particlesfromthedatasetscontainingFab21todetermineastructure
ofFimH-Fab824,asthesedatasetscontainedalargepoolofparticles
withgoodorientationdistributionfordataprocessing(Supplementary
Fig.5).ThevolumesresultingfromtheseanalysesshowedFimHinthe
inactive conformation (Fig. 1B, G, Supplementary Figs. 5, 6). Even
though the sample pool contained either Fab475 or α-methyl-man-
nose,theintermediateandfinalvolumesofthesestructuresdonot
contain data supporting the placement of a ligand in the pocket
(SupplementaryFig.15H–K).
Together,themodelsprovideastructuralrationaleforthepre-
viousobservationthatmAb824canbindallconformationsofFimH
butpreventstheconformationaltransitionbetweenactiveandinac-
tiveconformations28.mAb824directlyinteractswithresiduesonFimH
that flip between the surface and core of the protein during
conformational transitions (Supplementary Fig. 1G). FimH residues
Leu34andVal35havebeenidentifiedasa“toggleswitch”inwhichthe
two side chains flip around the β-strand axis, essentially swapping
places with each other in the two conformational states28. In the
inactiveFimH-Fab824structure,Phe54fromFab824packsagainstthe
sidechainofLeu34whileVal35pointsintothecoreofFimH(Fig.3E).In
theactivestate,thesidechainofVal35isonthesurfaceofFimHand
packsagainstPhe54fromFab824(Fig.3F).Fab824alsomakescontacts
withthesidechainsofTyr108andLys76ontheadjacentstrandswithin
thesameβ-sheet(SupplementaryFig.15G).Threeepitoperesidueson
separate strands of the β-sheet, Val36, Leu107, and Val75, do not
interactwithFab824butpacktightlyagainsteachother.Together,the
packing of mAb824 and the β-strand residues pin the β-strands in
place,preventingtheformationofthe6-ÅgaprequiredfortheLeu34/
Val35switch.Thus,byinteractingwithalargesetofdynamicresidues
andpackingdirectlyagainstLeu34intheinactivestateoragainstVal35
intheactivestate,Fab824preventstherearrangementsrequiredfor
transitionbetweenthetwoconformations.

Methods
ExpressionandpurificationoffimbrialtipsfromE.coli
ThegenesencodingforFimC(withahis-tagattheC-terminus)and
FimF-FimG-FimH(FimHUniProtID:P08191)ofthefimbrialtipswere
clonedintopRSET-Bexpressionplasmidaspreviouslydescribed14.The
plasmid was transformed into BL21 (DE3) cells via electroporation,
platedonLBagar,andincubatedovernight.Thecellsweregrownat
37°C in 1L cultures until the optical density at 600nm (OD600)
reached0.5–0.7.Proteinexpressionwasinducedbytheadditionof
0.5mMIPTG,followedbyovernightgrowthat22°C.Cellswerehar-
vestedbycentrifugationandresuspendedinPBSbuffercontaining
5mMimidazole.ThecellswerelysedusingaFrenchpressat1000psi,
andthelysatewasclarifiedbycentrifugation.Theclarifiedlysatewas
loadedontoanickelcolumnandwashedwithPBSbuffercontaining
5mMimidazole.AsecondwashstepwasperformedwithPBSbuffer
containing50mMimidazole.ThefimbrialtipswereelutedwithPBS
buffer containing 100mM imidazole. The protein was then further
purifiedbysizeexclusionchromatographyusingaSuperdex75col-
umn. Fractions containing the fimbrial tips were pooled and
concentrated.

GenerationofmonoclonalantibodiesandFabfragments
The lectin domain of FimH was used to raise the monoclonal anti-
bodiesaspreviouslyreported56andthisstudyusedfrozenhybridoma
stocksfromthispreviousstudytogeneratetheantibodies.ThemAbs
were expressed and purified from hybridoma cellcultures.The cell
cultures were clarified by centrifugation, and the supernatant was
loadedontoaProteinG-agarosecolumn.Thecolumnwaswashedwith
IgGbindingbuffer,andthemAbswereelutedwithIgGelutionbuffer
fromThermoFisherScientific.ThemAbswerefurtherpurifiedbysize
exclusionchromatographyusingaSuperdex200columninPBSbuf-
fer.PurifiedmAbswerethendigestedtoFabfragmentsusingagarose-
immobilized ficin (Thermo Fisher Scientific) and purified by size-
exclusionchromatographyusingaSuperdex75column.PurifiedFab
fragmentsweretestedforthepresenceofresidualFcfragmentsby
ELISAusinggoatanti-mouseIgG(Fc-specific;Sigma-Aldrich),following
themanufacturer’sinstructions.

Fimbrialtip-Fabcomplexassembly
All protein concentrations were measured at UV 280nm using a
NanoDrop.Thefimbrialtips/Fabcomplexeswereassembledatamolar
ratioof1:1.2,whereeachFabisaddedtoamolarconcentration1.2×the
molarconcentrationofthefimbrialtips.Thisratioensuredmaximal
Fab occupancy of FimH while minimizing background seen in test
micrographsarisingfromunboundFabs(SupplementaryFig.3A).

Cryo-electronmicroscopy
Proteinsampleswereassembledattheratiolistedaboveandatthe
concentrations listed in Table 1. Protein solutions were applied to
glow-dischargedgrids(CF-ProtochipsInc.;ANTcryo–SingleParticle,
LLC; Quantifoil, SPT Labtech), blotted, and plunge-frozen in liquid
ethaneusingamanualplungingapparatusatroomtemperature.Data
collectionwasperformedusinganFEITitanKriostransmissionelec-
tronmicroscopeoperatingat300kV(equippedwithaGatanimage
filter(GIF)andpost-GIFGatanK3Summitdirectelectrondetector)or
an FEI Glacios (equipped with a Gatan K3 Summit direct electron
detector) using the software packages Leginon (v3.5) or SerialEM
(v4.1)57,58.

Dataprocessing
Movies were aligned, corrected for beam-induced motion, dose-
weighted, and binned (2× – if collected in super-resolution mode)
using Patch Motion Correction in cryoSPARC(v4.4.1) orcryoSPARC
Live59. CTF parameters were estimated using Patch CTF59. For the
datasets FimH+Fab824/926, FimH+Fab21/824, and FimH+Fab21/
475/824, micrographs were excluded if they fell outside of these
thresholds:−0.1to−2.5nmdefocus;aCTFfitof2–6;andarelativeice
thickness of 1.050–1.250. For datasets FimH+Fab475/824 and
FimH+Fab824/926,particleswerepickedusingBlobPickerwiththese
parameters:minimumandmaximumparticlediametersof100–300Å
withaseparationdistanceof0.4diametersusinganellipticalblob.For
datasetsFimH+Fab21/824andFimH+Fab21/475/824,particleswere
template picked with templates generated from a low-resolution
volumeofFab21/475/824.Binnedparticles(4×)andtworoundsof2D
Classificationwereusedtoremovenoiseandpoorlyaligningparticles,
andtheremainingparticleswereusedinabinitioReconstructionand
3D Homogenous Refinement. Particles were re-extracted with
decreasedbinning(1.3×),andadditionalroundsof3Drefinementwere
completed,includingHomogenousRefinement,Non-UniformRefine-
ment,andLocalRefinement.Datasetsweresentthrougharoundof3D
ClassificationwithhardclassificationandmasksasshowninSupple-
mentary Fig. 2, followed by additional 3D refinement with an FSC
cutoffof0.143.ForthestructureFimH+Fab824,particlecoordinates
from datasets FimH+Fab21/475/824 and FimH+Fab21/824 were
exported to Relion (v5.0) after 3D Classification and Non-Uniform
RefinementincryoSPARC.Particleswerere-extractedinRelion,used
toreconstructastartingvolume,andrefinedusingRelionRefine3D
applyingBlushRegularization60asdepictedinSupplementaryFig.4.
Thefinalrefined,unsharpenedhalfmapsforalldatasetswereexported
toPhenix(v1.20)wheredensitymodificationandadditionalresolution
estimation was performed (FSC cutoff of 0.5)61,62. Local Resolution
EstimationwasperformedincryoSPARCusingthemapsgeneratedin
thefinalroundofrefinementandmappedontothedensitymodified
volumeinChimeraX(v1.7and1.9)63.ConicalFSCswerecalculatedin
cryoSPARCusingtheOrientationDiagnosticstool64,65.

Modelbuilding
PDB ID 3JWN and 15C8 were used as initial models for FimH and
Fab475, Fab824, and Fab92614,66. The FimH sequence was manually
updatedinmodel3JWNandFabsequenceswerethreadedonto15C8
usingtheModellerfunctioninChimeraX67.AlphaFold3wasusedto
generate an initial model of Fab2168. The model from the dataset
FimH+Fab826/926(PDBID9ME5)wasusedastheinitialmodelfor
FimH + Fab824. The cryo-EM volume information for Fab475 was
combined with the known glycosylation pathways of antibodies in
mammals69,70, known atypical glycosylations seen on a subset of
humanantibodyvariableloops41,42,theknownligandsofFimH5,6,9,and
the sugars identified via mass spectrometry in this manuscript and
usedtoinfertheidentityofthesugarsplacesintheglycanatN90.
ModelswereiterativelyrefinedusingacombinationofISOLDE(v1.7)in
ChimeraX (v1.7), Coot (v0.9.8.7), and phenix.real_space_refine
phenix.validation_cryoeminPhenix(v1.20)62,63,71–73.Thephenix.valida-
tion_cryoemtoolwasusedtogeneratemodelstatistics.

Imagegenerationandcalculationsformicrographsand
proteinmodels
CryoelectronmicrographsweremotioncorrectedusingPatchMotion
CorrectionincryoSPARCorcryoSPARCLive(v4.4.1)andGaussianfil-
teredandcontrastadjustedinFiji(2.14.0/1.54f).ChimeraX(v1.7)was
usedfor:volumealignment(FittoModeltoolorthe“fit”command),
modelalignment(Matchmakertoolorthe“align”command),buried
surface area calculations (“measure buriedarea” command), hydro-
phobicity calculations (“hydrophobic coloring” tool), and hydrogen
bonds identification (“hbond” tool). Models and volumes were ren-
deredinChimeraX.Allelectronmicrographimage,volume,andpro-
teinmodelfigureswereassembledinAdobeIllustratorCC6(v26.0.1).

Glycopeptidesampleprocessing
EachoftheFabmolecules(N=1)waspreparedseparatelyinparallel.
Each aliquot of individual Fabs (5µg) was prepared in 200mM Tris
buffersolution,wassubjectedtoa5-minuteheatshockat95°C,was
reduced with 5mM tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) for 30min at 60°C, and was alkylated with 20mM chlor-
oacetamide, followed by a 15-min quench using TCEP. Sodium
deoxycholatewasaddedtoafinalconcentrationof1%.Proteinswere
digestedovernightat37°Cwitheithertrypsin(1:50μg/μgprotease:-
protein) (Promega) or Chymotrypsin (1:70µg/µg protease:protein)
(Promega). The SDC in all samples was precipitated twice with 2%
formic acid (FA), after which samples were desalted using Strata-X
33µm 10mg/1mL Polymeric Reversed Phase SPE cartridges (Phe-
nomenex)byconditioningthecartridgewith1mLacetonitrile(ACN)
followedby1mL0.2%formicacid(FA)inwater.Peptideswerethen
loadedontothecartridge,followedbya1mLwashwith0.2%FAin
water74.Peptideswereelutedwith400μLof0.2%FAin80%ACNand
driedviavacuumcentrifugation.Individualtrypsinorchymotrypsin
digestionswererepeatedforeachFabinparallel.Thesesampleswere
furtherprocessedusingaPNGaseFtreatment,wheresampleswere
heat-shocked for 5min at 95°C to inactivate proteases before they
wereincubatedwith500unitsofPNGaseF(NewEnglandBiolabs)for
4h at 37°C. Following PNGase F treatment, these samples were
desalted using the same protocol as above and dried via vacuum
centrifugation. After drying, all samples werestored at−70°C until
massspectrometryanalysis.

Glycopeptidedataacquisition
Glycopeptides were analyzed on an Orbitrap Ascend Tribrid Mass
Spectrometer (Thermo Fisher Scientific – Orbitrap Ascend Tune
Application 4.2.4321) coupled to a Vanquish Neo UHPLC (Thermo
Fisher Scientific) using the Orbitrap Ascend Tune Application
(v4.2.4321).First,sampleswerereconstitutedin0.1%FAandloadedon
aC18trapcolumn(300µm×5mm,5µmparticles,PepMapNeo)using
a 300nL/min flow using buffer A (0.1% formic acid). Subsequently,
sampleswerelinearlyelutedusinga55-mingradientrangingfrom2.2%
bufferB(99.9%acetonitrilewith0.1%FA)to28%bufferBonaC18
analytical column (75µm×25cm, 1.7µm particles, IonOptics). The
columnwaswashedfor5minwith99%Bafterseparationandequili-
bratedwith100%bufferA.Precursorswereionizedusingananospray
flexionizationsource(ThermoFisherScientific)heldat+2.0kVcom-
paredtoground,andtheinletcapillarytemperaturewasheldat275°C.
Theinstrumentmethodcollectedspectrainadata-dependentfashion
usingfullscan(MS1)settingsof120,000resolvingpowerat200m/z,a
normalizedAGCtargetof250%(1,000,000charges),andanMS1mass
rangefrom400to1800m/z.Dynamicexclusionwassetat15s,and
onlycharges2–6wereselectedforfragmentation.Precursorionswere
isolatedforMS/MSscansusinga0.7m/zquadrupoleisolationwidth,
and measuredintheOrbitrapwithanMS2resolutionof30,000at200m/
z, a normalized AGC target set to 200% (100,000 charges), and a
maximuminjectiontimeof59ms.Selectedprecursorionswerefrag-
mented with stepped collision energy/higher-energy collisional dis-
sociation (sceHCD) performed using 20, 30, and 40% normalized
collision energies. sceHCD scans containing at least four glycan-
specific oxonium ions (126.055, 138.0549, 144.0655, 168.0654,
186.076, 204.0865,274.0921, 292.1027,and 366.1395m/z) triggered
electron-transfer/higher-energy collision dissociation (EThcD) scans,
where precursor ion isolation was performed with the quadrupole
using a 1.6m/z isolation window, an AGC target of 200% (100,000
charges), anamaximuminjectiontimeof251ms.EThcDfragmenta-
tion was performed using a 50ms reaction time and a 2e6 reagent
target.

PNGaseF-treatedpeptidedataacquisition
ThePNGaseF-treatedglycopeptides(N=1)wereanalyzedusingthe
sameLC-MSsetupandgradientastheglycopeptides.Theinstrument
methodusedanMS1resolutionof120kat200m/z,anormalizedAGC
target of 100% (400,000 charges), and an MS1 mass range ranging
from 375 to 1600m/z. Dynamicexclusion was set at 30s, and only
charges2–6wereselectedforfragmentation.HCDMS/MSscanswere
performedusinga0.7m/zquadrupoleisolationwidth,andfragment
ionsweremass-analyzedintheOrbitrapwithanMS2resolutionof15k
at200m/z.HCDMS/MSscansusedanormalizedAGCtargetsetto
100% (50,000 charges), a maximum injection time of 27ms, and a
normalizedcollisionalenergyof30%.

Glycopeptidedataanalysis
Raw data were searched using Byonic (v5.5.2)75. Each glycopeptide
searchforN-linkedglycosylationwasconductedseparately,witheach
search using the Fab-specific FASTA sequence file. The N-glycan
databaseconsistedof309uniquemammalianN-glycancompositions.
Carbamidomethylationwassetasafixedmodification,andoxidation
onmethionine,deamidationonN,andacetylationontheN-terminus
were used as variable modifications. A maximum of 3 miscleavages
wereallowed,andamaximumofthreeglycositeswereallowedforany
one glycopeptide. Glycan quantificationwasperformed on the MS1
levelusingSkyline76.DataweregraphedusingR(version4.4.0).Fil-
teringmetricsincludedaByonicscoregreaterthanorequalto200,
andalogProbvaluegreaterthanorequalto2.
O-Pair Search was performed in MetaMorpheus (v1.1.1)77,78. The
‘Glyco Search’ option was selected, and the O-glycopeptide search
feature and the O-glycan were enabled. Gdb glycan database was
selected.The‘keeptopNcandidates’featurewassetto50,andData
TypewassetasHCDwithchildscandissociationsetasEThcD.The
‘maximumO-glycanallowed’wassettofiveunlessotherwisenoted.In
silicodigestionparametersweresettogeneratedecoyproteinsusing
reversed sequences,andtheinitiatormethioninefeature wassetto
‘variable’.Themaximummodificationisoformsallowedwas1024,and
theminimumandmaximumpeptidelengthvaluesweresetto5and
60,respectively.Numberofmissedcleavageswassetto3.Precursor
andproductmasstoleranceswere10and20ppm,respectively,and
theminimumscoreallowedwasthree.Modificationsweresetascar-
bamidomethylonCasfixed,oxidationonM,andN-terminalacetyla-
tionasavariable.
All identified glycopeptide spectra were manually verified for
accurateidentification.Weonlyincludedidentifications:aftermanual
verification with at least a 50% sequence coverage after removalof
unreliablefragmentannotations(e.g.,toolargemasserror,c-orz-ions
n-terminal of proline, or improbable isotope cluster), with reliable
glycanassignment(e.g.,possibleglycancompositions,ornomultiple
fucosylationwithhighNeu5Acoxoniumsignal),andconvincingoxo-
niumionsignalcomparedtotheglycancomposition(e.g.,>10%ofTIC
isoxoniumion-derived,Neu5Gcdiagnosticionsfoundwhencompo-
sitionhasNeu5Gc).Identificationsalsousedglycan-lossfragmentsand
HexNAc-onlyresiduestostrengthenclaimsontheglycancomposition.

SamplepreparationfornativeMS
EachunprocessedFabsample(N=1),containing10μgoftheprotein,
was buffer-exchanged into 150mM aqueous ultrapure ammonium
acetate (pH 7.2) by ultrafiltration (Millipore Amicon) with a 10kDa
cutofffilter.Theproteinconcentrationwasadjustedto2–3μMpriorto
nativemassspectrometryanalysis.

NativeMSanalysis
SampleswereanalyzedusinganOrbitrapAscendTribridMassSpec-
trometer(ThermoFisherScientific–OrbitrapAscendTuneApplica-
tion4.2.4321)operatinginintactproteinmode.Sampleswereionized
usingborosilicatenanoelectrosprayemittersfabricatedin-housefrom
borosilicatecapillaries(0.78mminnerdiameter,1.00mmouterdia-
meter)(SutterInstruments).Thesprayvoltagewassetto1.5kV,source
fragmentation was 60V, source temperature was 300°C, and ions
weremassanalyzedintheOrbitrapwitharesolutionsetto30k(atm/z
200).EachFabwasmeasuredforatleast1mincontinuousacquisition.

NativeMSdataanalysis
The accurate masses of the Fabs were extracted by averaging and
deconvolutingtheelectrosprayionizationspectrumtoazero-charge
spectrumusingUniDec(v8.0.2)79.Thestandardsettingswereexcept
foranexpectedpeakof0.85Th,apointsmoothwidthsetto10,and
artifactsuppression(Beta)setto0.

DeglycosylationofmAbsandSDS-pagequantification
mAbdeglycosylationwasperformedwithEndoglycosidaseH(EndoH,
NEB#P0702)andEndoglycosidaseS(EndoS,NEB#P0741)according
tothemanufacturer’sprotocol.Briefly,20µgofmonoclonalantibody
were combined with 10X Buffer and 1µL of deglycosylase in total
volumeof20µL.Reactionwasperformedat37°Covernight.Halfof
thereactionwasanalyzedonSDS-PAGEforcompletionofthedegly-
cosylationreaction.

Antiserumadsorption
MicewereimmunizedwithpurifiedlectindomainofFimHfromE.coli
strainK12.Activityoftheobtainedantiserumwasdeterminedbyserial
dilutionsinELISAusing96-wellplatescoatedwithpurifiedFimH-FocH
fimbriae.Dilutionfactorthatistheclosesttothesaturationlevelof
chromogenicsignal(within1:5000–30,000rangedependingonthe
dayofexperiment)wasusedfortheadsorptionexperiments.Bacteria
expressing fimbriae with no FimH and with FimH-FocH or FimH-
FocHF1L variants were grown overnight, washed with PBS, con-
centratedtoOD650=9.0,andmixed1:1withthedilutedantiserain
Eppendorftubes.Adsorptionwasperformedbyrotatingthetubesat
16rpmatroomtemperaturefor1h.Uponcompletion,themixturewas
centrifuged at~9000×g for 3minand the supernatant wasfiltered
througha0.45µmmembraneandusedintheELISA.

Enzyme-linkedimmunosorbentassay
ELISAsforthemAb475deglycosylationandanti-FimHserumadsorp-
tionexperimentswereperformedascompletedpreviously28.Type1
pilicontainingthetipsidenticaltothoseusedforthecryo-EMrecon-
struction(FimHfromE.colistrainK12)wereexpressedbyandsheared
off the recombinant bacteria.Purified pili were immobilized onthe
surfaceofmicrotiterwells.Themonoclonalantibodiesorpolyclonal
serum wereaddedfor45minincubation.Thelevelofantibodybinding
wasdeterminedbyanti-mouseIgGsecondaryantibodiescoupledwith
horseradish peroxidase. The average of means obtained in three
independentexperimentsareplottedonthegraphswithcombined
standarddeviationshownaserrorbars,andP-valuescalculatedusinga
Student’sttestforpairwisecomparisontoevaluatethesignificance
independently(command“ttesti”inSTATAv14).

SequencealignmentsofmAb475-likeantibodies
OutgroupantibodysequenceswereidentifiedusingtheProteinBLAST
toolfromNCBI.ThelightchainsequenceofmAb475wasusedasthe
query.N-andC-terminalaminoacidsfromthemAb475-likeantibodies
were trimmed. MEGA7 was used to align sequences and build the
phylogenetictree80.Aftertheinitialalignment,thetreewastrimmedto
only include the branches containing at least one mAb475-like
antibody.

AntibodysequencingofV-regionofmAb21lightand
heavychains
TotalmRNAwasisolatedfromhybridomacellsusinganAllPrep®DNA/
RNAMinikit(Qiagen),followedbycDNAsynthesiswiththeiScript™
RT cDNA Synthesis Kit (BioRad) according to the manufacturer’s
procedure.TheV-regionswerePCRamplifiedusing2–3μLfromthe
25-μLRTreactionvolume.PrimersarelistedinSupplementaryTable7.
Gel-purified PCR products were subjected to sequencing by
GenewizInc.

Modelpreparationformoleculardynamicssimulations
Forsimplicityandtoreducethecomputationalcost,FimHwastrun-
catedafterresidue160,keepingonlythelectindomain(LD).Fab926
wastruncated after residues 115 (heavychain) and 112 (light chain).
Fab475wastruncatedafterresidues118(heavychain)and106(light
chain).Fab824wastruncatedafterresidues117(heavychain)and107
(lightchain).Fab21wastruncatedafterresidues125(heavychain)and
106(lightchain).Theseresiduesfallintheloopthatformsthebridge
betweenthetwoβ-sandwiches(Igfolds)thatmakeupeachheavyand
lightchain.TheC-terminalresidueinthetruncationisatleast6Åaway
from every residue except for the neighboring residue in the poly-
peptidechain,andvisualinspectiondemonstratesthatthereareno
side chain contacts between the N-terminal and C-terminal β-sand-
wiches. After truncation, the resulting C-terminus was capped with
N-methylamidetoneutralizeitscharge.Allhistidinesweresettobe
protonatedattheside-chainNεatom.

Moleculardynamics
Molecular dynamics (MD) simulations were performed using the
complexesdeterminedthroughcryo-EM.Briefly,thesimulationswere
performed with the program NAMD(v2.12)81 using the all-hydrogen
CHARMM22forcefield82withtheCMAPextension83,84andtheTIP3P
modelofwater.Theproteinwassolvatedusingacubicwaterboxwith
sidelengthof118Å(mAb926),125Å(mAb475),100Å(mAb824),and
116Å(mAb21)andallwithperiodicboundaryconditions.Chlorideand
sodiumionswereaddedtoneutralizethesystemandapproximatea
saltconcentrationof150mM.Duringthesimulations,thetemperature
waskeptconstantat300Kor330KbyusingtheLangevinthermostat
withadampingcoefficientof1ps−1,whilethepressurewasheldcon-
stantat1atmbyapplyingapressurepiston85,86.Additionalparameters
canbefoundinSupplementaryTable5.First,a50-nslongsimulation
wasperformedat300Ktodeterminestableinter-domainsidechain
contactsbetweenLDandtheFabdomainsandthedomainswitheach
other.Thiswasfollowedbya50-nslongsimulationat330Ktoaccel-
eratesamplingofevents.P-valueswerecalculatedwithGraphpad.com.

Mannosefittingandminimization
To determine whether mannoseclashes with the 29–31 loop of the
Fab926heavychainwesuperimposedeachframeofthetrajectories
onto a previously published model of lectin domain in the inactive
conformationboundtomannose27,33.Thesuperimpositionwasdone
by aligning the Cα atoms of residues 1–6 and 44–48 of the lectin
domain followed by visual inspection of the resulting model. The
model minimization was performed with the program CHARMM
(v35b1)andconsistedof100stepsofsteepestdescentminimization
performedinvacuoand500stepsofconjugategradientminimization
performedinadielectriccontinuum.

Rigid-bodydocking
TheonlinetoolpyDockWEB87wasusedtoperformdockingofFab926
toaconformationofLDwithboundmannosewheretheclamploopis
closed,correspondingtotheactiveconformation(PDBcode1UWF)
andtoaconformationoflectindomainwheretheclamploopisopen
butmannoseisstillbound,correspondingtoasnapshotsampledafter
330ns of a 330-K simulation performed in reference33. PyDockWEB
performsprotein-proteinrigid-bodydockingusingelectrostaticsand
desolvationenergies.Mannosewasomittedfromthecalculationsince
pyDockWEBcanonlyperformprotein-proteindocking.

Distanceandvolumemeasurements
Pocketvolumes(boundbyvanderWaalssurfaces)wereidentifiedand
measuredinCASTpFold88forthecryo-EMmodelofFimH-Fab926,the
modelfromtheMDsimulation,andthemodelfromtheMDsimulation
andminimization.Thesolvent-accessiblesurfaceenclosedvolumeof
an α-methyl-mannose molecule was calculated using the “Measure
Volume and Area” tool in ChimeraX63. Inter-atom distances were
measuredusingVMD(v1.8.7).Mannosewasfitintoeachframeofthe
simulation of the model from MD simulation as described in the
Mannose Fitting methods section. The average pairwise inter-atom
distancesbetweenmannoseandFab926andbetweenmannoseand
FimHwerecalculatedbyaveragingdistancesoverthelast16nsofthe
fullsimulation,whenthegapbetweentheproteinsappears.

Figuregenerationandassembly
Plots were generated using RStudio (v4.4.0) or EXCEL (v16.101.1).
ProteinvolumeandmodelimagesweregeneratedinChimeraX(v1.7
and v1.9). All images and plots were assembled into panels using
AdobeIllustratorCC(v26.0.1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, Hvorecny et al. provide structural evidence for the molecular mechanism of antibody binding to the lectin\ndomains of the bacterial adhesin FimH. Notably, the cryoEM structures offer a molecular rationale for distinct antibody-\nmediated mechanisms previously characterized by the same senior authors. These findings are complemented by adhesion\nassays, mass spectrometry, and molecular dynamics simulations.\nOverall, this is an excellent manuscript with rigorous validation of each result using a diverse set of techniques. It offers an\nintriguing perspective on how these antibodies bind and regulate bacterial adhesion. However, I have some concerns\nregarding the novelty of the study. As noted, significant prior work has been conducted on these systems, and the primary\nadvances here are (1) the cryoEM structures—though some NMR data was already available for certain systems—and (2)\nthe demonstration that one antibody binds via a covalently attached glycan. I raise the question (without a definitive answer,\nas I have mixed feelings about it) of whether these findings meet the high novelty standards of this journal.\nAdditionally, I have several comments that could be addressed and discussed:\n1. The authors' presentation of the FimH binding mechanism is somewhat misleading. They propose that the allosteric\ntransition from a weak- to a strong-binding state is ligand-activated, which appears to contradict the commonly accepted\ncatch-bond mechanism (see Nat Commun 7, 10738 (2016)). In that model, shear flow triggers the transition by separating\nthe lectin and pilin domains. While ligand binding is a prerequisite—resulting in what that study termed the A_bound state—\nthe transition to the S_bound state is force-activated. Ligand binding alone only facilitates the A_free to A_bound transition.\nCould the authors clarify how their findings fit within this framework?\n2. The authors suggest that glycan removal from mAb475 leads to suppressed binding activity (Figure 2F). However, the\ndata indicate a more nuanced picture: the normalized binding response drops from 1 to 0.2, not zero. Unless this is due to\nnoise, it implies only a fivefold reduction in binding affinity, which corresponds to a relatively modest free-energy difference\n(~1–2 kcal/mol). Could the authors further discuss this result?\n3. On page 6, the authors describe MD simulations of FimH-Fab926 and conclude that the protein-protein interface is flexible\nenough to allow mannose binding. However, mannose is relatively bulky. Could they provide quantitative data on whether\nmannose could actually fit into the space between the two proteins?\n4. Relatedly, a recent study (Biophys J 122, 2744 (2023)) highlighted the role of dynamic fluctuations in the binding site in\ndistinguishing the binding behavior of pilin-bound versus isolated lectin. Based on their new structures, do the authors have\ninsights into how antibody binding might affect active site dynamics?\n5. Do the authors have cryoEM structures of FimH-Fab824 alone? This would be an important addition to their structural\ndataset. If the proposed mechanism of Fab824 is correct, it should bind to the inactive state of FimH in the absence of\nFab21. Is this observed?\n6. Regarding the proposed dynamic trapping mechanism, is there supporting data from flow-based assays? Specifically,\ndoes Fab824 prevent FimH from transitioning into the strong-binding state, thereby suppressing the catch-bond effect? It is\nunclear (apologies if I missed it) whether this was addressed in previous studies. If not, this would be valuable evidence to\nconfirm the proposed mechanism.\n7. Minor: Throughout the text, the authors refer to figure panels using lowercase letters, whereas the figures themselves use\nuppercase.", "reviewer_3": "This manuscript by Hvorecny et al describes in detail four mechanisms by which antibodies may inhibit binding of bacterial\nadhesion pili to target cells. Their detailed analysis, based on cryo-EM data and molecular dynamics simulations provides a\nbasis for informed design of vaccines and therapeutics.\nWhile some of the results have been published previously, the current data provide empirical support for previous and\ncurrent conclusions. The only stretch appears in the authors’ statements regarding a relationship between glycan-containing\nantibodies in this study and understanding the development of cancer. As this is not my field, perhaps the authors are\ncorrect, but more text would be needed to understand what they are trying to convey. More specifically:\nL373-378 \"Further mutations and the breaking of tolerance could generate cells capable of transforming into cancer. \"\nPlease explain 'breaking of tolerance' Also, state what is having 'Further mutations'. It appears that it is the antibody that has\n'hypervariable loop glycans', so how is that 'microbial selection’?\nMinor details:\nL101-103. \"The activating mAb21 acts as a molecular wedge between the lectin and pilin domains, while allowing FimH to\nretain flexibility at the lectin-pilin interface.\"\n-- I don't see how it can do both these things -- if it is a wedge, then it blocks flexibility.\nL 1109. the labeling is unclear -- rename 'lectin domain' to 'lectin binding pocket' - or if this is not true, then change the text to\ndefine this better. In panel E - this is not FimH-Fab926, it is Fab to Fab interactions.", "reviewer_4": "To the Authors,\nThis manuscript presents a compelling investigation into the mechanism of action of mAb475, proposing an elegant model\nof ligand mimicry through an N-linked glycan on its hypervariable loop to competitively inhibit FimH-mediated adhesion. The\nintroduction effectively establishes the context and significance of this work, highlighting the importance of understanding\nantibody-mediated disruption of bacterial adhesion for the development of novel antimicrobials. The potential for host\nantibodies to employ such a mechanism represents a noteworthy contribution to the field.\nHowever, my primary concern revolves around the quality and presentation of the mass spectrometry data supporting the\nidentification and characterization of the N-linked glycan on mAb475. Given the rarity of such glycosylation events in\nantibody variable regions, robust and unambiguous evidence is crucial. The current manuscript lacks clarity regarding the\nglycopeptide verification criteria and does not provide sufficient spectral evidence to support this central claim confidently.\nFurthermore, the characterization of identified glycans in Table SX is unclear, particularly regarding their derivation from the\nglycopeptide data. If Byonic software was used for glycopeptide identification, example annotated MS/MS spectra should be\nprovided to allow for proper evaluation of the data quality. These issues necessitate a major revision of the manuscript, with\nparticular attention to the \"Methods\" section and the representation of the mass spectrometry data. Detailed comments\noutlining specific areas for improvement are provided below.\nComment 1 -\nDiscrepancy between the cryo-EM data and the reported glycan analysis of Fab 475: In the results section (Line 138), you\nmention that the cryo-EM density within the glycan binding pocket accommodates five sugar residues from an N-linked\nglycan. However, the glycan profiling data presented for N-glycosylation at position 28 on the Fab 475 light chain (Extended\nData Fig. 6) indicates that sialylated and other complex glycans constitute a significant percentage of the total identified\nglycans. This observation requires further clarification. Could you please provide a more detailed explanation reconciling the\ncryo-EM structural interpretation with the diverse glycan population identified by mass spectrometry?\nFurthermore, I reiterate my previous concern regarding the characterization of the glycan composition presented in Table\nSX. Given your statement that Fab 475 exhibited highly heterogeneous spectra, precluding intact mass calculations, the\nassignment of specific glycan compositions in this table remains unclear. Please elaborate on the methodology used to\ndetermine these compositions in the absence of intact mass information. Specifically, if the glycan compositions were\ninferred from glycopeptide fragmentation data (e.g., using Byonic), this should be explicitly stated and supported by\nrepresentative annotated MS/MS spectra, as requested previously. The link between the observed cryo-EM density and the\nheterogeneous glycan population identified by mass spectrometry needs to be clearly established and justified.\nComment 2 -\nThe potential for contamination in the Fab preparations to influence the mass spectrometry results: The Author correctly\npoints out, Fab glycosylation is a relatively minor occurrence compared to the near-universal glycosylation of the Fc region.\nConsequently, even trace amounts of Fc contamination in your supposedly purified Fab samples could significantly skew\nthe glycosylation analysis and potentially lead to misinterpretation of the data. In the \"Methods\" section (Line 568), it was\ndescribe the expression and purification of the raised mAbs. However, it is crucial to detail how the purification efficiency\nwas rigorously assessed to confirm the purity of the Fab samples. Please provide specific information on the methods used\nto determine the extent of any potential contamination by intact IgGs, Fc fragments, other antibody variants, or\nnon-antibody proteins. Quantifying the purity of your Fab preparations is essential to ensure the reliability of\nglycosylation analysis.\nComment 3 -\nMy third comment concerns the database search strategy employed for glycopeptide analysis and the manual verification\nprocess. It was mentioned that performing the database search against only the Fab protein sequence. While this focuses\nthe search, Byonic software, like other search algorithms, can potentially assign oxonium ion-containing spectra to the\nprovided sequence even with limited backbone fragment ion (b- and y-ion series) evidence, potentially leading to false-\npositive glycopeptide identifications. Therefore, I strongly recommend performing a parallel database search that includes\ncommon background proteins, such as other immunoglobulin/IgG proteins, to assess the specificity of your glycopeptide\nidentifications. Furthermore, you must clearly articulate the specific strategies and factors considered during your manual\nverification of the glycopeptides. What criteria were used to distinguish true positives from potential false positives reported\nby Byonic? Providing details on the required number and type of fragment ions, glycan diagnostic ions, and any other quality\nmetrics used for manual validation is essential. Finally, to allow for independent evaluation of your findings, the raw data and\nthe complete Byonic search results file should be provided as supplementary material. This level of transparency is crucial\nfor verifying the robustness of the glycopeptide identifications.\nComment 4 -\nThe manuscript states that for the MD simulations, FimH was truncated after residue 160 (retaining only the lectin domain),\nand Fab926 heavy and light chains were truncated after residues 115 and 112, respectively. However, it is unclear whether\nmAb475, the central antibody in this study, was also subjected to truncation for the MD simulations. If mAb475 was indeed\ntruncated, please explicitly state the truncation points for its heavy and light chains. Furthermore, it is crucial to discuss the\npotential impact of these truncations – both for FimH and any truncated antibody – on the MD simulation analysis and the\nresulting conclusions. A discussion addressing the rationale for these truncations and their potential limitations or effects on\nthe simulation outcomes is necessary for a comprehensive understanding of your findings.\nComment 5 -\nThe stoichiometry used in the preparation of the fimbrial tips/Fab complexes for structural studies: In Line 577, you mention\nthat these complexes were generated from a mixture of fimbrial tips and Fab fragments at a 1:1.2 ratio. Please elaborate on\nthe rationale for choosing this specific ratio. Were other ratios explored? If so, what were the outcomes, and why was the\n1:1.2 ratio deemed optimal for generating the complexes used in your study? Providing this information will help clarify the\nexperimental design and the potential for different stoichiometries to influence the observed structures.\nComment 6 -\nGlycopeptide Sample Processing: In Line 627, you mention the use of both trypsin and chymotrypsin for protein digestion.\nHowever, it is unclear throughout the subsequent text which specific data originates from the chymotryptic digests. If data\nderived from chymotryptic digestion are presented or discussed in the manuscript, please explicitly state this to avoid\nconfusion. Regarding the peptide desalting step in Line 635, you mention using \"Strata-X cartridges (Phenomenex).\" To\nensure reproducibility and allow others to follow your methodology precisely, please specify the exact type of stationary\nphase used in these cartridges by providing the relevant catalog number. Different Strata-X phases possess varying\nproperties that can influence peptide recovery and selectivity.\nOther comments -\nSeveral inconsistencies in abbreviation usage and grammatical errors require careful revision throughout the manuscript.\nFor instance, the abbreviation for \"cryo-electron microscopy\" appears inconsistently as both \"cryoEM\" and \"cryo-EM\" (e.g.,\nLines 28 and 314, respectively). Please ensure consistent use of \"cryo-EM\" throughout the text and introduce the full term\nbefore its first abbreviation.\nHere are the comments presented as a point-wise list for the authors:\n• Abbreviations: Ensure consistency in the abbreviation \"cryo-EM\" throughout the text. Please use \"cryo-EM\" instead of\n\"cryoEM\" (e.g., Line 28, 109, 111, 136). Always use the full term before introducing an abbreviation for the first time.\n• Line 42: Remove the comma before \", cell attachment proteins\".\n• Line 46: Capitalize \"Gram-negative\".\n• Line 48: Remove the comma after \"rod-shaped,\".\n• Line 75: Change \"(mAb)\" to \"(mAbs)\".\n• Line 109: Add a full stop at the end of the sentence.\n• Line 113: The sentence \"Full fimbrial tips (FimH/G/F with chaperone FimC) complexed with multiple Fabs without\noverlapping epitopes increased the size of our target while allowing us to unambiguously identify each Fab bound to FimH\n(Figure 1a-d, Ext. Data Fig. 5a).\" is lengthy and could be broken down for improved readability.\n• Line 125: Add a hyphen to \"ligand-binding\".\n• Lines 323-331: Change \"beta strand\" to \"beta-strand\" and \"beta strands\" to \"beta-strands\".\n• Line 506: Add a full stop at the end of the sentence.\n• Line 557: Change \"...plated on LB agar overnight.\" to \"...plated on LB agar and incubated overnight.\"\n• Line 559: Consider revising to: \"...induced by the addition of 0.5 mM IPTG, followed by overnight growth at 22 °C.\"\n• Line 565: Change \"...size exclusion chromatography on a Superdex 75 column.\" to \"...size-exclusion chromatography\nusing a Superdex 75 column.\"\n• Line 573: Change \"...size exclusion chromatography on a Superdex 200 column in PBS buffer.\" to \"...size-exclusion\nchromatography using a Superdex 200 column in PBS buffer.\"\n• Line 574: Change \"...digested to Fab fragments using agarose-immobilized ficin from Thermo Fisher Scientific and purified\nby size exclusion on a Superdex 75 column.\" to \"...digested to Fab fragments using agarose-immobilized ficin (Thermo\nFisher Scientific) and purified by size-exclusion chromatography using a Superdex 75 column.\"\n• Line 578: Change \"...in a 1:1.2 ratio.\" to \"...at a 1:1.2 molar ratio.\"\n• Lines 596-597: Change \"100-300 Å min-max diameter\" to \"minimum and maximum particle diameters of 100-300 Å\".\n• Line 627: Change \"Glycopeptide sample processing:\" to \"Glycopeptide Sample Processing\".\n• Line 628: Change \"Each aliquot of individual Fabs contained 5 µg of the Fab molecule in 200 mM Tris buffer solution...\" to\n\"Each aliquot of individual Fabs (5 µg) was prepared in 200 mM Tris buffer solution...\"\n• Line 645: Change \"Glycopeptide data acquisition:\" to \"Glycopeptide Data Acquisition\".\n• Line 668: Change \"PNGase F-treated peptide data acquisition:\" to \"PNGase F-treated Peptide Data acquisition:\".\n• Line 676: Change \"Glycopeptide data analysis:\" to \"Glycopeptide Data Analysis:\".\n• Line 677: Consider revising to: \"...with each search using the Fab-specific FASTA sequence file.\"\n• Lines 686 and 688: Change \"Oglycan\" to \"O-glycan\".\n• Line 712: Change \"Mab\" to \"mAbs\".\n• Line 714: Change \"microgram\" to \"µg\" and \"microliter\" to \"µL\".\n• Line 716: Change \"Reaction was performed at 37oC overnight.\" to \"Reaction was performed at 37 °C overnight.\"\n• Line 718: Change \"Antiserum adsorption:\" to \"Antiserum Adsorption:\".\n• Line 725: Suggestion: \"Adsorption was performed by rotating the tubes at 16 rpm at room temperature for 1 h.\"\n• Line 726: Change \"Upon the completion\" to \"Upon completion\", \".45\" to \"0.45\", and \"uM\" to \"µm\".\n• Line 729: Change \"Enzyme-linked immunosorbent assay:\" to \"Enzyme-linked Immunosorbent Assay:\".\n• Lines 736-737: Correct \" T-test and \"ttesti\" to the appropriate statistical test notation (e.g., t-test).\n• Line 740: Change \"Sequence Alignments of mAB475-like Antibodies:\" to \"Sequence Alignments of mAb475-like\nAntibodies:\".\n• Line 746: Change \"V-Region\" to \"V-regions\" and \"manufacturer’s\" to \"the manufacturer's\".\n• Line 763: Change \"cryo EM\" to \"cryo-EM\".", "reviewer_author_response": "Thank you for your comprehensive reading of our manuscript. Below, we address the concerns raised during\nthe review process and have marked our responses with “>>” and italicized text.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this work, Hvorecny et al. provide structural evidence for the molecular mechanism of antibody binding to the\nlectin domains of the bacterial adhesin FimH. Notably, the cryoEM structures offer a molecular rationale for\ndistinct antibody-mediated mechanisms previously characterized by the same senior authors. These findings\nare complemented by adhesion assays, mass spectrometry, and molecular dynamics simulations.\nOverall, this is an excellent manuscript with rigorous validation of each result using a diverse set of techniques.\nIt offers an intriguing perspective on how these antibodies bind and regulate bacterial adhesion. However, I\nhave some concerns regarding the novelty of the study. As noted, significant prior work has been conducted\non these systems, and the primary advances here are (1) the cryoEM structures—though some NMR data was\nalready available for certain systems—and (2) the demonstration that one antibody binds via a covalently\nattached glycan. I raise the question (without a definitive answer, as I have mixed feelings about it) of whether\nthese findings meet the high novelty standards of this journal.\n>>The importance of FimH as a model allosteric protein and as a model adhesin has made it a\ncritical target and the focus of decades of study. We firmly believe that this work will be of use to\nbroad audiences in several fields and highlight our rationale here.\n● As the Reviewer notes, we demonstrate the previously unobserved, direct binding of a\nvariable-loop glycan into the ligand binding pocket of an adhesion from a pathogen.\n● This antibody provides an evolutionary rationale for variable-loop glycosylations in\nthe polyclonal antibody response.\n● The common use of adhesins by pathogens across evolution and the presence of ~15%\nvariable loop glycosylation in mammalian serum antibodies suggest this is a previously\nunidentified yet common, broadly elicited response to infections.\n● The direct interaction between an adhesin from a pathogen and hypervariable-loop\nglycosylations potentially links infection with the development and progression of\nseveral lymphomas and autoimmune disorders.\n● As the Reviewer notes, our full structures of the FimH-antibody interactions offer\nmolecular rationales for distinct mechanisms of antibody inhibition.\n● These four mechanisms have not previously been demonstrated for other bacterial\nadhesins or other similar allosteric proteins.\n● Complete structures of the FimH-Fab interactions also facilitate molecular dynamic\nsimulations which demonstrate the complex interplay of adhesins, ligands, and\nantibody inhibitors.\n● Crucially, the structures also provide the paratope side of the interaction, creating\nblueprints for inhibitors of FimH and other bacterial adhesins. Current work by our\ngroups is leveraging these structures in both the rational and de novo design of\ninhibitors.\nOur manuscript is an important contribution to the fields of immunology, microbiology, host-\npathogen interactions, carcinogenesis and cancer progression, inhibitor design, and vaccine\ndevelopment.\nAdditionally, I have several comments that could be addressed and discussed:\n1. The authors' presentation of the FimH binding mechanism is somewhat misleading. They propose that the\nallosteric transition from a weak- to a strong-binding state is ligand-activated, which appears to contradict the\ncommonly accepted catch-bond mechanism (see Nat Commun 7, 10738 (2016)). In that model, shear flow\ntriggers the transition by separating the lectin and pilin domains. While ligand binding is a prerequisite—\nresulting in what that study termed the A_bound state—the transition to the S_bound state is force-activated.\nLigand binding alone only facilitates the A_free to A_bound transition. Could the authors clarify how their\nfindings fit within this framework?\n>>Our presentation of the FimH binding mechanism was imprecise. We have updated the text\nto clarify our description of the FimH binding mechanism. In addition to Nat Comm 7, 10738\n(2016), we also refer to JACS 141, 936-944 (2019, Sauer et al), where the authors show that\nligand binding changes the thermodynamic cycle, shifting the equilibrium constant of domain\nseparation by a factor of 2000 toward the S-state in the absence of mechanical force. This\nechoes previous studies of mAb21, the S-state-specific antibody. mAb21 binding to full-length\nFimH is limited in the absence of mannose but binding is drastically increased in the presence\nof mannose. In the current study, we visualize mAb21 binding to the interdomain region of the\nlectin domain of full-length FimH in the S-state. In this sample condition, the S-state was\ninduced in presence of mannose but in the absence of mechanical force.\n>>We have updated the description of FimH in the introduction:\n“FimH also serves as a model allosteric protein as it undergoes dramatic\nconformational rearrangements that decrease ligand dissociation7,10. In the\nabsence of ligand, FimH adopts a compressed and inactive conformation\nstabilized by a fixed interface between the lectin and pilin domains, with the\nligand-binding pocket relaxed and open10 (Ext. Data Fig. 1C). Ligand binding\ncloses the pocket and shifts FimH into an intermediate conformation. In the\nintermediate conformation, the mannose-binding loops, N-terminus, and clamp\nloop encircle the ligand, but the lectin-pilin domain interface remains fixed11 (Ext.\nData Fig. 1D). Ligand binding also alters the equilibrium constant of lectin-pilin\ndomain separation in FimH11–13. This change increases the proportion of FimH\nthat adopts an elongated active conformation where the pilin and lectin domains\nare separated, although the domain-separated conformation of full length, ligand\nbound FimH is less populated in the absence of shear force (Ext. Data Fig. 1E).\nTensile mechanical force facilitates lectin-pilin domain separation, which pulls the\npilin domain away from the lectin domain. Separation stabilizes the active\nconformation and results in a force-enhanced catch-bond interaction between the\nligand and the pilin domain7,10,14,15.”\n>>We have also updated our introduction of orthosteric antibodies:\n“Orthosteric antibodies, like mAb475, directly compete for the ligand binding pocket\nand behave like classical competitive inhibitors27. mAb475 acts similarly to FimH\nligands; the core epitope includes mannose binding residues on FimH and the\nantibody triggers the closing of the ligand binding pocket and shifts the\nconformational equilibrium (Ext. Data Fig. 1F).”\n2. The authors suggest that glycan removal from mAb475 leads to suppressed binding activity (Figure 2F).\nHowever, the data indicate a more nuanced picture: the normalized binding response drops from 1 to 0.2, not\nzero. Unless this is due to noise, it implies only a fivefold reduction in binding affinity, which corresponds to a\nrelatively modest free-energy difference (~1–2 kcal/mol). Could the authors further discuss this result?\n>>The limitations of the ELISA complicate quantitative determinations of free-energy changes\nusing these data. However, these limitations likely cause the assay to underestimate the\nreduction in binding affinity for deglycosylated mAb475. The limitations include: the cleavage\nsites of EndoS and EndoH, the completeness of deglycosylation, and the non-linearity of the\nELISA data, as described below.\nNon-linearity of the assay: At low and high antibody concentrations, the ELISA loses linearity,\neffectively compressing the window of the assay. This is observed in a titration curve of mAb475\n(now Supp. Fig. 2). We can use this titration curve to account for the non-linearity of the assay\nand estimate the fold change of mAb475 binding. The 5-fold drop in the signal in Fig. 2C\ncorresponds to a 30-60 fold drop in mAb475 binding to FimH.\nCleavage sites of EndoS and EndoH: EndoS and EndoH do not remove all sugar moieties from\nthe modified asparagine, which likely affects the apparent binding observed in the assay. Both\nEndoH and EndoS cleave the β-1,4 linkage between the two GlcNAcs in the chitobiose core of\nan N-linked glycan, leaving behind GlcNAc1 on Asn90 and Asn28. The sugars remaining on\nAsn90 and Asn28 cause the deglycosylated light chain of mAb475 to migrate more slowly than\nthe light chains of the other mAbs (Supp Fig. 1; black arrows).\nImportantly for the binding assay, the remaining GlcNAc1 on Asn90 is involved in FimH\nbinding and likely contributes to the signal detected in the deglycosylated sample of the ELISA.\nThe moiety adopts an ordered position in the binding pocket when bound both as a sugar in the\nligand oligomannose-3 [Wellens et al, 2008 PLoS Pathogens] and as part of mAb475 [this\nstudy]. In addition to the hydrophobic interaction of with Thr51 of FimH [Wellens et al, 2008\nPLoS Pathogens], GlcNAc1 on mAb475 also interacts with Arg100 of the mAb475 heavy chain,\nsupporting the ordering of the arginine and promoting the Arg100 direct interaction with FimH.\nCompleteness of deglycosylation: Cleaving with EndoH and EndoS does not remove every\nglycan at Asn90, which shifts the baseline of the assay. Glycans remaining on Asn90 of\nmAb475 can bind FimH, generating signal in the assay. Asn90 sits in a recessed site on\nmAb475, in the groove between the two β-sandwiches (Ig folds) of the variable domains from\nthe heavy and light chain. Inefficiencies in processing of recessed glycans in variable loop\ndomains has been highlighted by others [Xiang et al. 2016 PNAS; Choiden et al. 2021 Blood].\nBased on the cleavage pattern with EndoH and EndoS, it is likely that the presence of the\nglycan at Asn28 also sterically interferes with cleavage of the glycan at Asn90, as is seen in\nglycan processing on HIV [Struwe et al. 2018 Cell Reports]. The site’s decreased accessibility\nmakes cleavage of the glycan less efficient, which is reflected in our SDS-PAGE of the\ndeglycosylated sample (Supp Fig. 1). In both gels, we see uncleaved light chain and species in\nthe mAb475 digests (white arrows) not found in the other antibody digests.\n>>We have updated the paragraph describing the deglycosylation and binding experiments as\nwell as the associated figures and figure legends.\n“We assessed the contributions of the glycans in mAb475 binding and inhibition\nof FimH by deglycosylating mAb475 and performing binding experiments (Figure\n2F, Ext. Data Fig. 9A). [...] Treatment with both Endo S and Endo H shifted the\nmajority of the mAb475 light chain closer to the predicted size of 25 kDa (Ext.\nData Fig. 9A, Supp Fig. 1) and decreased mAb475’s ability to bind FimH\n(Figure 2F, Ext. Data Fig. 9A). The pattern and multiple species in the mAb475\ndigestions with EndoS, EndoH, and EndoS/EndoH shows that we may not\nachieve full deglycosylation at Asn90 (Ext. Data Fig. 9A, Supp Fig. 1).\nCombined with the non-linearity of the ELISA assay, the assay limitations likely\nlead to an underestimation of the magnitude of glycan contribution to antibody\nbinding (Supp Fig. 2). Along with the mass spectrometry and cryo-EM results,\nthe biochemical results confirm that the complex glycans primarily found on\nAsn28 do not contribute to FimH binding while the high-mannose and hybrid\nglycans on Asn90 drive binding and inhibition by mAb475.”\nSupplementary Figure 1:\n“Supplementary Figure 1. SDS-PAGE gels of endoglycosidase-treated\nmAbs. The sugars remaining on Asn90 and Asn28 cause the deglycosylated\nlight chain of mAb475 to migrate more slowly than the light chains of the other\nmAbs (black arrows). The mAb475 digests (white arrows) contain uncleaved light\nchain and species not found in the other antibody digests.”\n“Supplementary Figure 2. Titrations of mAb475 in ELISA assays against FimH-\ncontaining pili. Red arrow indicates the concentration used for the ELISA data\nshown in Fig. 2C and Ext. Data Fig. 9A. N = 4 technical replicates per titration;\nerror bars = ± S.D..”\n3. On page 6, the authors describe MD simulations of FimH-Fab926 and conclude that the protein-protein\ninterface is flexible enough to allow mannose binding. However, mannose is relatively bulky. Could they\nprovide quantitative data on whether mannose could actually fit into the space between the two proteins?\n>>Our analyses indicate that mannose can fit into the space between the two proteins. We\nquantitatively evaluated the fit of mannose in the protein-protein interface between FimH and\nFab926 at two points: 1. using the model from the MD simulation in which mannose was not\nincluded, and 2. using the model from the MD simulation after a minimization procedure in\nwhich mannose was added.\nModel from MD simulation: To evaluate whether mannose would fit into the pocket between\nthe FimH lectin domain and Fab926 observed in the MD simulations, we calculated atom-atom\ndistances and the enclosed pocket volume. We docked mannose into the binding pocket for\neach frame of the simulation as described in the methods (Ext. Data Fig. 11C). The pocket\ncalculator CASTpFold did not detect any space between FimH and Fab926 in the cryo-EM\nmodel. In contrast, the model from the 50ns time point in the simulation contains a pocket with a\ntotal volume of 194 Å3 (calculated from van der Waals surface in CASTpFold). An α-methyl-\nmannose molecule encloses a volume of 149 Å3 (calculated from solvent excluded surface in\nChimeraX). We also calculated the average pairwise inter-atom distances between mannose\nand Fab926 and between mannose and FimH by averaging distances over the last 16 ns of the\nfull simulation, when the gap between the proteins appears (Ext. Data Fig. 11F). Calculated\ndistances, both averages and maximum in the time range, are listed in Table 3. Some atom-\natom distances between FimH and mannose remain closer than van der Waals radii. Together,\nthis suggests that there is enough space for mannose to fit in the observed pocket, but that\nwithout mannose present in the simulation, FimH has not adopted a “mannose interacting”\nconformation. Clashes are not unexpected given that mannose was not included in the\nsimulation and rigid-body fit into the model.\nAverage\nMaximum Average Maximum\nHeavy\nAverage All Atom Average All\nModel Interface Atom\nHeavy Atom Distance Atom\nDistance\nDistance (Å) (Å ± S.D.) Distance (Å)\n(Å ± S.D.)\nFab926-Mannose 2.1 ± 0.4 3.5 1.1 ± 0.4 2.6\nFimH-Fab926\nMD Simulation\nFimH-Mannose 1.9 ± 0.3 2.6 1.1 ± 0.3 2.0\nFab926-Mannose 2.9 N/A 1.8 N/A\nFimH-Fab926\nMD Simulation\n+ Minimization\nFimH-Mannose 2.7 N/A 1.7 N/A\nTable 3: Pairwise inter-atom distances between Fab926 and mannose or FimH and\nmannose. The full description of the calculations can be found in the methods.\nMinimized model from MD simulation: We reasoned that minimal movements would be\nneeded to fully accommodate mannose in the pocket between FimH and Fab926. To test this,\nwe performed minimization after docking mannose in the last frame of the simulation, as\ndetailed in the methods. Comparison between the models before and after the minimization\nrevealed a heavy-atom RMSD of 0.7 Å and an all-atom RMSD of 0.8 Å, indicating the models\nremained highly similar. The pocket from the minimized model contains a total volume of 263\nÅ3, easily accommodating the α-methyl-mannose volume. Calculated distances are listed in\nTable 3. Heavy-atom and all-atom pairwise distances increased and hydrogen bonds between\nmannose and residues D54, D47, Q133 and the N-terminus of FimH were formed as is seen in\nexperimentally determined structures containing mannose.\n>>We have updated the text and figures to include these quantitative analyses.\n“[...] Together, these conformational changes create space for mannose in the\nbinding pocket while mAb926 is still bound to FimH, highlighting the flexibility\nwithin the FimH-mAb926 binding interface (Ext. Data Fig. 11C). Minimization of\nthe model from 50 ns time point of the simulation while including a-methyl\nmannose shows the molecule forming the appropriate hydrogen bonds and\nintermolecular distances (heavy atom Fab926-Mannose distance: 2.9 Å; heavy\natom FimH-Mannose distance: 2.7 Å) within a pocket volume (263 Å3) large\nenough to accommodate the molecule volume (149 Å3) with minimal deviation\nfrom the simulation (heavy atom RMSD: 0.7 Å, all-atom RMSD: 0.8 Å) (Fig. 2J,\nExt. Data Fig. 11C and G, Table 3). Our simulations suggest that mannose may\nbind FimH while Fab926 is bound, and Fab926 may bind when mannose is\nbound, with each destabilizing the binding of the other.”\n“Ext. Data Fig. 11. Molecular dynamics simulations suggest flexibility at the\nFimH-Fab926 binding interface. [...] C. Slice through the molecular dynamics\nsimulation at 50 ns (left) and the molecular dynamics simulation at 50 ns with\nminimization (right); FimH (dark blue surface) bound to Fab926 (orange surface),\nwith mannose shown in green. [...] G. Heavy atom distances between the docked\nmannose and Fab926 during the 50 ns simulation. The gap between FimH and\nFab926 widened after 34 ns of simulation time (red vertical arrow).”\n4. Relatedly, a recent study (Biophys J 122, 2744 (2023)) highlighted the role of dynamic fluctuations in the\nbinding site in distinguishing the binding behavior of pilin-bound versus isolated lectin. Based on their new\nstructures, do the authors have insights into how antibody binding might affect active site dynamics?\n>>Our simulations show that each antibody primarily affects the dynamics at the location where\nthe antibody binds. For simplicity and to reduce computational costs, only a single antibody was\nincluded in each simulation and we truncated FimH and antibody chains as described in the\nmethods. We performed two, 50-ns long simulations at 300 K in the absence of mannose for\neach of the FimH-antibody pairs. We calculated the Cα RMSD across the trajectories for the\nclamp loop (residues 2-22 [Interlandi et al; Proteins 2016]) and for the swing loop (residues 23-\n32 [Interlandi et al; Proteins 2016]) for each simulation and compared each antibody-bound\nFimH structure to its unbound parallel. The results are shown in Ext. Data Table 3.\nMD Simulation P-value MD Simulation P-value\nFimH\nAntibody Clamp Loop (antibody-bound Swing Loop (antibody-bound\nConformation\nRMSD ± SEM [Å] vs. unbound) RMSD ± SEM [Å] vs. unbound)\nActive\n(High Affinity) None 0.84 ± 0.01 N/A 0.62 ± 0.06 N/A\n+ Mannose\nActive\nmAb475 0.58 ± 0.03 0.0114 1.33 ± 0.10 0.0255\n(High Affinity)\nActive\nNone 1.69 ± 0.44 N/A 0.55 ± 0.01 N/A\n(High Affinity)\nActive\nmAb21 0.96 ± 0.27 0.3115 0.44 ± 0.06 0.0997\n(High Affinity)\nActive\nmAb824 0.90 ± 0.01 0.2545 0.50 ± 0.01 0.0403\n(High Affinity)\nInactive\nNone 1.46 ± 0.01 N/A 0.64 ± 0.08 N/A\n(Low Affinity)\nInactive\nmAb824 1.16 ± 0.03 0.012 0.80 ± 0.15 0.4255\n(Low Affinity)\nInactive\nmAb926 1.09 ± 0.05 0.0161 0.89 ± 0.15 0.2747\n(Low Affinity)\nExt. Data Table 3: RMSD Calculations for the FimH lectin domain in the absence and presence\nof each mAb. N = 2-3 simulations; The P-values were calculated from a two-tailed Student's t-test; P-\nvalues < 0.05 highlighted in bold. The full description of the calculations can be found in the methods.\nClamp loop: Both mAb475 and mAb926 interact with the clamp loop and stabilize the loop, as\nindicated by a decrease in RMSD, when bound to the FimH conformation observed in the cryo-\nEM structures. However, mAb475 stabilizes the clamp loop in active (high-affinity) conformation,\nwhile mAb926 and mAb824 stabilize the clamp loop of FimH in the inactive (low affinity)\nconformation. In contrast, neither mAb824 nor mAb21 show a statistically significant change in\nstability of the clamp loop in the active conformation. It is important to note that the clamp\nsegment in the active (high affinity) conformation is generally much more stable than in inactive\n(low affinity) conformation [Interlandi et al; Proteins 2016].\nSwing loop: mAb475 showed a statistically significant change in swing loop dynamics, as\nmeasured by an increase in RMSD when compared to the lectin domain alone. The increase in\ndynamics fits with previous work showing that ligand binding changes the thermodynamic cycle\nand alters the equilibrium constants between FimH conformations [2016 Sauer et al. Nat.\nComm; 2019 Sauer et al JACS]. mAb824 shows a small but significant increase on the stability\nof the swing loop in the active conformation.\n>>We have updated the text to include these results.\n>>Fab475 and Fab926:\n“[...] Both mAb475 and mAb926 interact with the clamp loop and stabilize the\nloop when bound to the FimH conformation observed in the cryo-EM structures\n(Ext. Data Table 3). mAb475 also showed a statistically significant change in\nswing loop dynamics. This increase fits with previous work showing that ligand\nbinding changes the thermodynamic cycle and alters the equilibrium constants\nbetween FimH conformationss7,11.”\n>>Fab21:\n“[...] The [mAb21] binds in between the FimH lectin and pilin domains but does\nnot specifically interact with the pilin domain (Ext. Data Fig. 6B). Our simulations\ndo not show a statistically significant change in loop flexibility when mAb21 is\nbound; however, this could be due to the limited time scale of the simulations\nperformed here35 (Ext. Data Table 3). [...]”\n>>Fab824:\n“Our simulations show that Fab824 stabilizes the clamp loop of FimH in the\ninactive conformation (Ext. Data Table 3). In contrast, mAb824 does not show a\nstatistically significant difference in stability on the active clamp loop in the limited\ntime scale of our simulations, though the decrease in RMSD trends towards\nstabilization. It is important to note that the clamp segment in the active\nconformation is generally much more stable than in inactive conformation33.\nThese results agree with our previous hypotheses that mAb824 stabilizes the\nconformation of FimH that it binds.”\n5. Do the authors have cryoEM structures of FimH-Fab824 alone? This would be an important addition to their\nstructural dataset. If the proposed mechanism of Fab824 is correct, it should bind to the inactive state of FimH\nin the absence of Fab21. Is this observed?\n>>In the absence of bound Fab21 or bound mannose, FimH-Fab824 adopts the inactive\nconformation. Additional data processing identified a pool of particles in three of our cryo-EM\ndatasets containing the FimH-Fab824 only complex. We pooled these particles where possible\nand determined a ~3.1 Å structure of FimH-Fab824, which has now been deposited in the PDB\nand EMDB with the IDs 9PTM and EMD-71842, respectively.\n>>We have added a description of this structure to the results and included method descriptions\nand additional figures to demonstrate our data processing scheme and analyses.\n>>Results section:\n“When bound solely to Fab824, FimH adopts the inactive conformation, which\nsupports the proposed mAb824 mechanism. In the cryo-EM datasets containing\ncombinations of Fab21, Fab475, Fab824, and α-methyl-mannose, we observe\ntwo-dimensional class averages containing FimH and Fab824 (Ext. Data Fig.\n3H). We used the particles from the datasets containing Fab21 to determine a\nstructure of FimH-Fab824, as these datasets contained a large pool of particles\nwith good orientation distribution for data processing (Ext. Data Fig. 4). The\nvolumes resulting from these analyses showed FimH in the inactive conformation\n(Fig. 1B and G, Ext. Data Fig. 4-5). Even though the sample pool contained\neither Fab475 or α-methyl-mannose, the intermediate and final volumes of these\nstructures do not contain data supporting the placement of a ligand in the pocket\n(Ext. Data Fig. 10H-K).”\n>>Methods section:\n“Data Processing: [...] For the structure FimH + Fab824, particle coordinates from\ndatasets FimH + Fab21/475/824 and FimH + Fab21/824 were exported to Relion\nafter 3D Classification and Non-Uniform Refinement in cryoSPARC. Particles\nwere re-extracted in Relion, used to reconstruct a starting volume, and refined\nusing Relion Refine3D applying Blush Regularization59 as depicted in Ext. Data\nFig. 4. The final refined, unsharpened half maps for all datasets were exported to\nPhenix (v1.20) where density modification and additional resolution estimation\nwas performed (FSC cutoff of 0.5)60,61. [...]”\n“Model Building: [...] The model from the dataset FimH + Fab826/926 (PDB ID\n9ME5) was used as the initial model for FimH + Fab824. [...]”\n>>Figures:\n“Ext. Data Fig. 3: Micrographs and two-dimensional class averages from FimH +\nFab datasets. [...] H. Representative class averages from the three datasets listed\nshowing one viewing direction of FimH bound to Fab824 only.”\n“Ext. Data Fig. 4: Data processing scheme and FSC curves for FimH + Fab824\nonly. Sample preparation and data collection strategy for FimH-Fab824 complex.\nBottom Right: Fourier Shell Correlation curves for the final maps generated in Relion\nand the density modified maps generated in Phenix. Pale colors represent Relion gold-\nstandard maps with an FSC cutoff of 0.143 (grey dash-dot line) and dark colors\nrepresent density modified maps with an FSC cutoff of 0.5 (black dashed line). Yellow\ncurves represent the full FimH-Fab structures with solvent mask only, while blue curves\nare reconstructions assembled using the focus mask shown in Ext. Data Fig. 2B.”\n“Ext. Data Fig. 10. Supplementary figure panels for FimH in complex with Fab926\nand Fab824. [...] H-K. Volumes and model of ligand binding pocket of FimH bound to\nFab824 only. Mannose position approximated by aligning mannose binding loops of the\nmodel containing FimH + Fab21/824/α-methyl mannose to the model containing FimH +\nFab824 only. H. Final, full, density-modified volume. I. Final, locally refined, density-\nmodified volume. J. Intermediate volume from FimH + Fab21/475/824 dataset. K.\nIntermediate volume from FimH + Fab21/824/ α-methyl mannose.”\n6. Regarding the proposed dynamic trapping mechanism, is there supporting data from flow-based assays?\nSpecifically, does Fab824 prevent FimH from transitioning into the strong-binding state, thereby suppressing\nthe catch-bond effect? It is unclear (apologies if I missed it) whether this was addressed in previous studies. If\nnot, this would be valuable evidence to confirm the proposed mechanism.\n>>Yes, there is published supporting data for the dynamic trapping mechanism in various assays. In\nKisiela and Magala et al (2021, PLoS Pathogens), we tested dynamic trapping using bio-layer\ninterferometry (BLI), binding of mAb21, and a test of cell adhesive properties of FimH under shear\nconditions.\nBLI: We found that pretreatment of FimH with Fab824 decreases binding of FimH to RNase B (a high-\nmannose containing protein) to the levels of FimH in the presence of saturating mannose (mannose\ncompetitively the inhibits binding of FimH to RNase B). This suggests that Fab824 locks FimH in the\ninactive conformation preventing FimH from productively engaging with RNase B.\nBinding of mAb21: We showed that pretreatment with mAb824 prevents binding of the active-state\nspecific antibody (mAb21) to FimH when mannose is added. As above, this demonstrates that inactive\nFimH in complex with mAb824 is unable to convert to the active state.\nShear conditions: Under dynamic rocking conditions, bacteria expressing FimH bind to red blood cells\nthrough surface glycans and engage the catch bond mechanism, causing the cells to aggregate. In\ncontrast, bacteria expressing FimH and pre-treated with Fab824 do not cause red blood cell\naggregation. This shows that Fab824, binding FimH in the inactive conformation, prevents FimH from\nproductively engaging with the glycans, likely by preventing the transition to the active, domain\nseparated state where the catch bond has been engaged.\n>>We have added a sentence in the introduction to address this point:\n“Dynasteric antibodies, (mAb824), arrest the conformational dynamics of FimH;\nthe antibodies bind both active and inactive conformations of FimH, but prevent\nthe transition between conformations28. mAb824 binding of the inactive\nconformation prevents FimH from productively engaging with glycans, even\nunder shear stress28. The core epitope for mAb824 is distal to the ligand-binding\npocket (Ext. Data Fig. 1F-G). ”\n7. Minor: Throughout the text, the authors refer to figure panels using lowercase letters, whereas the figures\nthemselves use uppercase.\n>>All references to figure panels have been updated to uppercase letters.\nReviewer #2 (Remarks to the Author):\nThis manuscript by Hvorecny et al describes in detail four mechanisms by which antibodies may inhibit binding\nof bacterial adhesion pili to target cells. Their detailed analysis, based on cryo-EM data and molecular\ndynamics simulations provides a basis for informed design of vaccines and therapeutics.\nWhile some of the results have been published previously, the current data provide empirical support for\nprevious and current conclusions. The only stretch appears in the authors’ statements regarding a relationship\nbetween glycan-containing antibodies in this study and understanding the development of cancer. As this is not\nmy field, perhaps the authors are correct, but more text would be needed to understand what they are trying to\nconvey.\n>>Please see our revised text below and in the manuscript.\nMore specifically: L373-378 \"Further mutations and the breaking of tolerance could generate cells capable of\ntransforming into cancer.\" Please explain 'breaking of tolerance' Also, state what is having 'Further mutations'.\nIt appears that it is the antibody that has 'hypervariable loop glycans', so how is that 'microbial selection’?\n>>Our explanation was unclear as previously written. We have updated the discussion:\n“Several B-cell malignancies are characterized by high-mannose or mixed type\nglycosylations on the hypervariable loops (CDRs) of Fabs produced from IgGs or\nB-cell receptors of these cells39–41. Specifically, the glycosylated position on\nmAb475 (Asn90) has previously been identified on a Fab from the B-cell receptor\nof lymphocytes from a patient with diffuse large B-cell lymphoma (Ext. Data Fig.\n6G)41,42. N-linked glycosylations on hypervariable loops have also been\ndescribed in follicular lymphoma and Burkitt and Burkitt’s lymphoma39–41. Microbial lectins,\nincluding adhesins found on E. coli, can interact with the B-cell receptors\ncontaining high-mannose type glycans, contributing to tumor cell persistence and\ngrowth36,37,43. Our data directly visualize the interaction between a glycosylated\nFab and a bacterial lectin, supporting the hypothesis that microbial lectins may\ncontribute to the progression of a subset of B-cell lymphomas.\n“Antigens from infectious microbes can also trigger the development of\nlymphomas and contribute to autoimmune disorders by stimulating B-cells\nreceptors44–47. While high-mannose glycans on hypervariable loops have\npreviously been associated with a subset of B-cell malignancies, it is not known\nwhat triggers the development of hypervariable loop glycosylation in these\nmalignancies37,41,48. mAb475 and the mAb475-like antibodies were elicited\nthrough a polyclonal antibody response to the FimH lectin domain24. This work\nshows that high-mannose glycans on B-cell receptors may be selected during an\ninfection by a microbe that uses mannose-binding lectins as part of its repertoire.\nThis finding supports the hypothesis that FimH and other mannose-binding\nlectins may trigger initial steps in the development of B-cell malignancies like\ndiffuse large B-cell lymphomas that present with high-mannose glycans on B-cell\nreceptors.”\nMinor details:\nL101-103. \"The activating mAb21 acts as a molecular wedge between the lectin and pilin domains, while\nallowing FimH to retain flexibility at the lectin-pilin interface.\"\n-- I don't see how it can do both these things -- if it is a wedge, then it blocks flexibility.\n>>We have updated the text to clarify our explanation of “molecular wedge.”\n“The activating mAb21 acts as a molecular wedge between the lectin and pilin\ndomains, preventing the fixed lectin-pilin interface from reforming.”\nL 1109. the labeling is unclear -- rename 'lectin domain' to 'lectin binding pocket' - or if this is not true, then\nchange the text to define this better. In panel E - this is not FimH-Fab926, it is Fab to Fab interactions.\n>>We have updated the figure and the legend to clarify what is presented in Ext. Data Fig. 9C-E.\n“Ext. Data Fig. 11. Molecular dynamics simulations suggest flexibility at the\nFimH-Fab926 binding interface. [...] D-E. Time series of side chain contacts\nformed between FimH and the variable domains of Fab926. The frames in which\na contact is formed in the simulation is indicated in cyan. Simulation was\ncompleted with the entire FimH lectin domain (residues 1-160) and Fab926\nheavy (2-115) and light chains (2-112), but only those residues that contact\nFab926 are shown in the plots. D. FimH-Fab926, light chain contacts. E. FimH-\nFab926, heavy chain contacts. F. Time series of side chain contacts formed\nbetween the heavy chain and the light chain of Fab926. The frames in which a\ncontact is formed in the simulation are indicated in cyan. [...]”\nReviewer #3 (Remarks to the Author):\nTo the Authors,\nThis manuscript presents a compelling investigation into the mechanism of action of mAb475, proposing an\nelegant model of ligand mimicry through an N-linked glycan on its hypervariable loop to competitively inhibit\nFimH-mediated adhesion. The introduction effectively establishes the context and significance of this work,\nhighlighting the importance of understanding antibody-mediated disruption of bacterial adhesion for the\ndevelopment of novel antimicrobials. The potential for host antibodies to employ such a mechanism represents\na noteworthy contribution to the field.\nHowever, my primary concern revolves around the quality and presentation of the mass spectrometry data\nsupporting the identification and characterization of the N-linked glycan on mAb475. Given the rarity of such\nglycosylation events in antibody variable regions, robust and unambiguous evidence is crucial. The current\nmanuscript lacks clarity regarding the glycopeptide verification criteria and does not provide sufficient spectral\nevidence to support this central claim confidently. Furthermore, the characterization of identified glycans in\nTable SX is unclear, particularly regarding their derivation from the glycopeptide data. If Byonic software was\nused for glycopeptide identification, example annotated MS/MS spectra should be provided to allow for proper\nevaluation of the data quality. These issues necessitate a major revision of the manuscript, with\nparticular attention to the \"Methods\" section and the representation of the mass spectrometry data. Detailed comments\noutlining specific areas for improvement are provided below.\n>>We have revised the results and methods to address the reviewers concerns below and have\nincluded and emphasized additional evidence and rationales in our determination of the\nglycosylations on mAb475.\nComment 1 -\nDiscrepancy between the cryo-EM data and the reported glycan analysis of Fab 475: In the results section\n(Line 138), you mention that the cryo-EM density within the glycan binding pocket accommodates five sugar\nresidues from an N-linked glycan. However, the glycan profiling data presented for N-glycosylation at position\n28 on the Fab 475 light chain (Extended Data Fig. 6) indicates that sialylated and other complex glycans\nconstitute a significant percentage of the total identified glycans. This observation requires further clarification.\nCould you please provide a more detailed explanation reconciling the cryo-EM structural interpretation with the\ndiverse glycan population identified by mass spectrometry?\n>>The mass spectrometry data agrees with the cryo-EM data. Fab475 contains two\nglycosylated asparagines: N28 and N90. Both are observed in the cryo-EM volume and show\nglycosylation by mass spectrometry; however, only the glycan at N90 is directly involved in the\ninteraction with FimH.\nThere is no evidence from the cryo-EM dataset that the glycan at N28 interacts with FimH. The\nglycan population identified by mass spectrometry at this residue demonstrates that N28 is\nglycosylated by a mixture of sialylated species (Ext. Data Fig. 7A). The N28 glycan is observed\nin the 2D class averages and in the filtered 3D map of the cryo-EM data; however, the map is\nnot detailed enough to fit a specific glycan (Ext. Data Fig. 7B). The flexibility of the glycan\ncombined with a mixture of species at this position produce a cryo-EM map that shows evidence\nof a covalently linked modification at N28 but that remains too poorly resolved to accurately\nbuild a glycan.\nExt. Data Fig. 3: Micrographs and two-dimensional class averages from\nFimH + Fab datasets. […] G. Two-dimensional class average of FimH +\nFab21/475. The glycan attached to Asn28 is visible as a hazy spot in the\naverage.\nExt. Data Fig. 7: Quantification of N-glycosylation 28 on the Fab 475 light\nchain and intact glycopeptide mass spectrometry analysis of the Fabs. A.\nFab molecules were subjected to intact glycopeptide mass spectrometry\nanalysis, and glycan abundances were quantified based on the area under the\ncurve of the extracted ion chromatogram. B. Volume of FimH + Fab475/824 with\nsite of Asn28 shown by white box. Inset: Low-pass filtered volume of FimH in\ncomplex with Fab475 showing volume for glycosylation at Asn28. [...]\nIn contrast, five sugars from the glycan attached to N90 are in fixed positions directly interacting\nwith FimH. The cryo-EM map is well resolved and we can extract some conformational details\nabout the glycan: A, the sugars in the 1 and 2 position show evidence of modifications to the\ncore ring; B, the 3 sugar does not contain the modifications in 1 and 2 but is connected to two\nadditional sugars in addition to its connection to the 2 sugar; C, mannose and oligomannose-3\nfrom previously determined structures of FimH fit the map of the sugar in the pocket very well;\nD, the volume of the 4’ sugar is less well-resolved, but does not show strong evidence for\nmodifications that are found in the 1 and 2 sugars; and E, filtering the volume to lower resolution\nshows that the 4’ sugar has some connecting volume suggesting two additional sugars in the\nglycan. Combining our cryo-EM data with the known glycosylation pathways of antibodies in\nmammals, atypical glycosylations seen on a subset of human antibody variable loops, the\nknown binding partners of FimH, and the sugars identified in Fig. 2C via mass spectrometry, we\nreason that the portion of the glycan resolved in the structure contains the five sugars shown in\nFig. 2B: 1 - GlcNAc, 2 - GlcNAc, 3 - Man, 4 - Man, 4’ - Man.\n>>We have updated the manuscript text to clarify the differences between N28 and N90:\n\"To identify and quantify the glycosylations on the Fabs of each antibody, we\ndigested the Fabs with either trypsin or chymotrypsin and analyzed the resulting\nintact glycopeptides using mass spectrometry on Fabs from each antibody\n(Figure 2C, Ext. Data Fig. 7-8). Only Fab475 exhibited N-glycosylation on its\nlight chain, with two glycosylated sites present in the light chain CDRs (Table 2).\nThe first site, Asn90 in CDR3, directly interacts with FimH as seen in the cryo-EM\ndata. Mass spectrometry identified only three glycan compositions attached to\nthe asparagine (Figure 2C). Two of these are high-mannose glycans, and the\nthird is likely a hybrid sialylated glycan. The second site, Asn28 in CDR1, does\nnot interact with FimH. Asn28 showed a diverse portfolio of N-linked glycans;\nhowever, over 95% of the glycan intensity consisted of sialylated glycans (Ext.\nData Fig. 7A). Inspection of the two-dimensional class averages and the cryo-\nEM volume surrounding Asn28 showed additional disordered volume in\nagreement with the mass spectrometry data (Ext. Data Fig. 3G and 7B).\nInvestigation of deamidated asparagines generated by PNGase F treatment\ncorroborates the identified glycans and the lack of N-glycosylation on the other\nFabs (Ext. Data Table 2). Further confirmation of glycosylation on Fab475 and\nthe absence of glycosylation on the other Fabs was obtained through native\nmass spectrometry analysis. Intact analysis of Fab475 revealed highly\nheterogeneous spectra that eluded simple deconvolution, which is characteristic\nof glycosylated proteins (Ext. Data Fig. 7C). In contrast, Fab926, Fab824, and\nFab21 revealed less complex and interpretable spectra with no indication of\nglycosylation (Ext. Data Fig. 7D-F).”\n>>We have updated the methods to provide rationale for the glycan built at N90:\n“Model Building: [...] The cryo-EM volume information for Fab475 was combined\nwith the known glycosylation pathways of antibodies in mammals63,64, known\natypical glycosylations seen on a subset of human antibody variable loops38,39,\nthe known ligands of FimH5,6,9, and the sugars identified via mass spectrometry\nand glycosidase digest in this manuscript to infer the identity of the sugars placed\nin the glycan at N90.”\nFurthermore, I reiterate my previous concern regarding the characterization of the glycan composition\npresented in Table SX. Given your statement that Fab 475 exhibited highly heterogeneous spectra, precluding\nintact mass calculations, the assignment of specific glycan compositions in this table remains unclear. Please\nelaborate on the methodology used to determine these compositions in the absence of intact mass information.\nSpecifically, if the glycan compositions were inferred from glycopeptide fragmentation data (e.g., using Byonic),\nthis should be explicitly stated and supported by representative annotated MS/MS spectra, as requested\npreviously. The link between the observed cryo-EM density and the heterogeneous glycan population identified\nby mass spectrometry needs to be clearly established and justified.\n>>We thank the reviewer for their careful consideration, as we agree that these are important\ndetails. To clarify, our data consist of both bottom-up (proteolytically derived glycopeptides) and\nintact mass (non-digested Fabs) glycoproteomic data. The statement about highly\nheterogeneous spectra for Fab475 was in relation to its intact mass data, which is indicative of\nglycosylation in the first place because of glycosite microheterogeneity. For bottom-up\nglycoproteomics data, glycopeptides were identified using Byonic searches that were specific to\neach Fab (i.e., using the FASTA sequence file specific to the Fab). The N-glycan database\nconsisted of 309 unique mammalian N-glycan compositions. Carbamidomethylation was set as\na fixed modification, and oxidation on M, deamidation on N, and acetylation on the N-term were\nused as variable modifications. A maximum of 3 miscleavages were allowed, and a maximum of\nthree glycosites were allowed for any one glycopeptide. Only glycopeptides were considered\nwith a Byonic score greater than or equal to 200, and a logProb value greater than or equal to 2.\nThe quantification was performed by extracting the MS1 XIC using Skyline.\n>>We edited the text (former lines 156:157) to further clarify that intact glycopeptides were used\nfor glycan quantification:\n“To identify and quantify the glycosylations on the Fabs of each antibody, we\ndigested the Fabs with either trypsin or chymotrypsin and analyzed the resulting\nintact glycopeptides using mass spectrometry.”\n>>We also strongly agree with the importance of annotated spectra for reliable confirmation of\nglycopeptide identifications. All annotated spectra are available at the repository as the Byonic\noutput is uploaded as listed in the Data Availability Statement (dataset identifier PXD057683):\n“The mass spectrometry proteomics data have been deposited to the\nProteomeXchange Consortium via the PRIDE partner repository with the dataset\nidentifier PXD057683 (Username: reviewer_pxd057683@ebi.ac.uk; Password:\nNcMsYBw7jsAA).”\n>>To improve accessibility, we have added the annotated spectra from the glycopeptides as\nSupplementary Data 1 and 2. Only identifications were included after manual verification with\nat least a 50% sequence coverage after removal of unreliable fragment annotations (e.g., too\nlarge mass error, c- or z-ions n-terminal of proline, or improbable isotope cluster), with reliable\nglycan assignment (e.g., possible glycan compositions, or no multiple fucosylation with high\nNeu5Ac oxonium signal), and convincing oxonium ion signal compared to the glycan\ncomposition (e.g., >10% of TIC is oxonium ion-derived, Neu5Gc diagnostic ions found when\ncomposition has Neu5Gc). Identifications also used glycan-loss fragments and HexNAc-only\nresidues to strengthen claims on the glycan composition. We have updated the methods to\ninclude this information; please see the response to Comment 3.\n>>As noted above, five sugars from the glycan attached to N90 are in fixed positions directly\ninteracting with FimH and the volume is resolved which allows us to place sugars in the model.\nPlease refer to the first part of Comment 1 for the full logic scheme of assigning the sugars\nplaced in the cryo-EM volume.\nComment 2 -\nThe potential for contamination in the Fab preparations to influence the mass spectrometry results: The Author\ncorrectly points out, Fab glycosylation is a relatively minor occurrence compared to the near-universal\nglycosylation of the Fc region. Consequently, even trace amounts of Fc contamination in your supposedly\npurified Fab samples could significantly skew the glycosylation analysis and potentially lead to\nmisinterpretation of the data. In the \"Methods\" section (Line 568), it was described the expression and\npurification of the raised mAbs. However, it is crucial to detail how the purification efficiency was rigorously\nassessed to confirm the purity of the Fab samples. Please provide specific information on the methods used\nto determine the extent of any potential contamination by intact IgGs, Fc fragments, other antibody variants, or\nnon-antibody proteins. Quantifying the purity of your Fab preparations is essential to ensure the reliability of\nglycosylation analysis.\n>>To address this concern, we performed a second search of the bottom-up mass spectrometry\ndata against the whole mouse proteome to confirm the purity of the Fabs. Quantification of the\nproteins was performed using the unique peptides identified for each protein. Fab475, Fab824,\nand Fab926 exhibit high purity and primarily contain keratin as a source of contamination (a\ncommon background contaminant in all proteomics experiments). In the Fab21 sample, only\nImmunoglobulin heavy constant gamma 2B (Ighgb2) was also identified at a similar\nabundance.\nAdditionally, identifications of the Fab475 glycans are performed at the glycopeptide level,\nwhere the glycan and the amino acid sequence are identified with high confidence. The amino\nacid sequences identified for the intact glycopeptides are: ASQNVSNVAWYQQKPGQSPK, and\nFCQQNSSFPF. A blast search against the full mouse proteome reveals that these are only\nidentical to the Fab475. The identified glycans are thus localized to the Fab475 and not to\nbackground proteins.\nWe have added this data as Ext. Data Fig. 8:\nExt. Data Fig. 8: Fab purity by Mass Spectrometry. A-D. Search of the bottom-up\nmass spectrometry data against the whole mouse proteome to confirm the purity of the\nFabs. Quantification of the proteins was performed using the unique peptides identified\nfor each protein. A (Fab475), B (Fab824), and C (Fab926) exhibit high purity and\nprimarily contain keratin as a source of contamination (a common background\ncontaminant in all proteomics experiments). In the Fab21 sample (D), only\nImmunoglobulin heavy constant gamma 2B (Ighgb2) was also identified at a similar\nabundance.\nComment 3 -\nMy third comment concerns the database search strategy employed for glycopeptide analysis and the manual\nverification process. It was mentioned that performing the database search against only the Fab protein\nsequence. While this focuses the search, Byonic software, like other search algorithms, can potentially assign\noxonium ion-containing spectra to the provided sequence even with limited backbone fragment ion (b- and y-\nion series) evidence, potentially leading to false-positive glycopeptide identifications. Therefore, I strongly\nrecommend performing a parallel database search that includes common background proteins, such as other\nimmunoglobulin/IgG proteins, to assess the specificity of your glycopeptide identifications. Furthermore, you\nmust clearly articulate the specific strategies and factors considered during your manual verification of the\nglycopeptides. What criteria were used to distinguish true positives from potential false positives reported by\nByonic? Providing details on the required number and type of fragment ions, glycan diagnostic ions, and any\nother quality metrics used for manual validation is essential. Finally, to allow for independent evaluation of your\nfindings, the raw data and the complete Byonic search results file should be provided as supplementary\nmaterial. This level of transparency is crucial for verifying the robustness of the glycopeptide identifications.\n>>These details are critical, as the reviewer points out. We have addressed some of these\nconcerns in responses to other comments, but to reiterate, we first investigated whether the\nidentified glycopeptides could be the result of contaminants in the sample by searching against\nthe entire mouse proteome, including other immunoglobulins such as IgG. However, we did not\nfind any hits for our identified glycopeptides against any other proteins, indicating that the\nidentified glycopeptides are Fab-derived rather than contaminant proteins. The Byonic results\nare uploaded to the PRIDE repository (shared in the Data Availability Statement section, please\nsee Comment 1 Response), allowing users to verify all spectra. Additionally, we provide\nannotated spectra in a PDF to increase accessibility to the key spectra (Supplementary Data 2\nand 3).\n>>The following snippet was added to the methods to provide additional details on the\nverification:\n“Only identifications were included after manual verification with at least a 50%\nsequence coverage after removal of unreliable fragment annotations (e.g., too\nlarge mass error, c- or z-ions n-terminal of proline, or improbable isotope cluster),\nwith reliable glycan assignment (e.g., possible glycan compositions, or no\nmultiple fucosylation with high Neu5Ac oxonium signal), and convincing oxonium\nion signal compared to the glycan composition (e.g., >10% of TIC is oxonium ion-\nderived, Neu5Gc diagnostic ions found when composition has Neu5Gc).\nIdentifications also used glycan-loss fragments and HexNAc-only residues to\nstrengthen claims on the glycan composition.”\nComment 4 -\nThe manuscript states that for the MD simulations, FimH was truncated after residue 160 (retaining only the\nlectin domain), and Fab926 heavy and light chains were truncated after residues 115 and 112, respectively.\nHowever, it is unclear whether mAb475, the central antibody in this study, was also subjected to truncation for\nthe MD simulations. If mAb475 was indeed truncated, please explicitly state the truncation points for its heavy\nand light chains. Furthermore, it is crucial to discuss the potential impact of these truncations – both for FimH\nand any truncated antibody – on the MD simulation analysis and the resulting conclusions. A discussion\naddressing the rationale for these truncations and their potential limitations or effects on the simulation\noutcomes is necessary for a comprehensive understanding of your findings.\n>>For simplicity and to reduce the computational cost, FimH and both chains of Fab475 and\nFab926 were truncated to single domains. Both chains of Fab475 and Fab926 were truncated in\nthe loop that forms the bridge between the two β-sandwiches (Ig folds) that make up each\nheavy and light chain, with the variable domain (N-terminus) being retained for the simulation.\nFab475 was truncated after residues 118 (heavy chain) and 106 (light chain). The C-terminal\nresidue in the truncation is at least 6 Å away from every residue except for the neighboring\nresidue in the polypeptide chain, and visual inspection demonstrates that there are no side\nchain contacts between the N-terminal and C-terminal β-sandwiches. We have added these\ndetails to the methods:\n“Model Preparation for Molecular Dynamics Simulations: For simplicity and to\nreduce the computational cost, FimH was truncated after residue 160, keeping\nonly the lectin domain (LD). Fab926 was truncated after residues 115 (heavy\nchain) and 112 (light chain). Fab475 was truncated after residues 118 (heavy\nchain) and 106 (light chain). Fab824 was truncated after residues 117 (heavy\nchain) and 107 (light chain). Fab21 was truncated after residues 125 (heavy\nchain) and 106 (light chain). These residues fall in the loop that forms the bridge\nbetween the two β-sandwiches (Ig folds) that make up each heavy and light\nchain. The C-terminal residue in the truncation is at least 6 Å away from every\nresidue except for the neighboring residue in the polypeptide chain, and visual\ninspection demonstrates that there are no side chain contacts between the N-\nterminal and C-terminal β-sandwiches.”\n>>We have also added text outlining our rational to the results section of the manuscript:\n“To explore the flexibility of Fab926 binding, we used molecular dynamics\nsimulations. We performed simulations using either the FimH-Fab926 structure\nor the FimH-Fab475 structure to determine the range of flexibility for each\ninteraction (Ext. Data Fig. 11-12). We trimmed computational costs by limiting\nour simulations to the lectin domain of FimH and the N-terminal variable domain\nβ-sandwich from the heavy and light chains on each Fab. Previous studies have\nshown that truncation of FimH after residue 160 has minimal effects on the\ndynamics of the lectin domain in the time frame of the simulations32,33. The\npersistence of side chain contacts between the β-sandwiches of the heavy chain\nand the light chain of the Fab interface over the course of the simulation indicate\nthe stability of the models (Ext. Data Fig. 11F).”\nComment 5 -\nThe stoichiometry used in the preparation of the fimbrial tips/Fab complexes for structural studies: In Line 577,\nyou mention that these complexes were generated from a mixture of fimbrial tips and Fab fragments at a 1:1.2\nratio. Please elaborate on the rationale for choosing this specific ratio. Were other ratios explored? If so, what\nwere the outcomes, and why was the 1:1.2 ratio deemed optimal for generating the complexes used in your\nstudy? Providing this information will help clarify the experimental design and the potential for different\nstoichiometries to influence the observed structures.\n>>We reasoned that a 1:1 molar ratio of fimbrial tips per individual Fab may leave some fimbrial\ntips undecorated at equilibrium, which could be worsened with any sample impurities from\nprotein purification or Fab generation and any inaccuracies in protein concentration\ndetermination. Initial screening using negative stain electron microscopy and a 1:3 molar ratio of\nfimbrial tip with a single Fab showed a high level of background when compared to fimbrial tips\nalone. Subsequent screening using a 1:1.2 molar ratio of fimbrial tips and multiple Fabs, where\neach Fab was added to a final concentration 1.2x the fimbrial tip concentration, produced\ncleaner negative stain micrographs, so we moved forward with cryo samples using this ratio\n(Ext. Data Fig. 3A).\nWe have adjusted the methods to clarify our choice of sample concentration and included\nexample negative stain micrographs.\n“Fimbrial Tip-Fab Complex Assembly: All protein concentrations were measured\nat UV 280 nm using a NanoDrop. The fimbrial tips/Fab complexes were\nassembled at a molar ratio of 1:1.2, where each Fab is added to a molar\nconcentration 1.2 x the molar concentration of the fimbrial tips. This ratio ensured\nmaximal Fab occupancy of FimH while minimizing background arising from\nunbound Fabs in test micrographs (Ext. Data Fig. 3A).”\nComment 6 -\nGlycopeptide Sample Processing: In Line 627, you mention the use of both trypsin and chymotrypsin for\nprotein digestion. However, it is unclear throughout the subsequent text which specific data originates from the\nchymotryptic digests. If data derived from chymotryptic digestion are presented or discussed in the manuscript,\nplease explicitly state this to avoid confusion. Regarding the peptide desalting step in Line 635, you mention\nusing \"Strata-X cartridges (Phenomenex).\" To ensure reproducibility and allow others to follow your\nmethodology precisely, please specify the exact type of stationary phase used in these cartridges by providing\nthe relevant catalog number. Different Strata-X phases possess varying properties that can influence peptide\nrecovery and selectivity.\n>>Peptides resulting from tryptic digestion were used to quantify N28 glycosylation on Fab475,\nand peptides derived from chymotrypsin for N90 glycosylation on Fab475. The peptides used\nfor quantification, along with the protease that generated them, are listed in Table 2 to clarify\nthis.\nAmino Peptide Sequence Protease Glycan Quantification\nAcid\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK NeuGc(1)\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK NeuGc(1)\nWYQQKPGQSPK\nWYQQKPGQSPK\nWYQQKPGQSPK\nTable 2: Glycan quantification on the Fab475 light chain. Both modified\namino acids are asparagines. Fab475 was digested, subjected to mass\nspectrometry analysis, and all identified glycans were quantified and validated as\ndetailed in the methods.\n>>The missing details on the desalting procedure have been added to the Strata-X methods\nsection:\n“The SDC in all samples was precipitated twice with 2% formic acid (FA), after\nwhich samples were desalted using Strata-X 33 µm 10 mg / 1 mL Polymeric\nReversed Phase SPE cartridges (Phenomenex) by conditioning the cartridge\nwith 1 mL acetonitrile (ACN) followed by 1 mL 0.2% formic acid (FA) in water.\nPeptides were then loaded onto the cartridge, followed by a 1 mL wash with\n0.2% FA in water. Peptides were eluted with 400 μL of 0.2% FA in 80% ACN\nand dried via vacuum centrifugation.”\nOther comments -\nSeveral inconsistencies in abbreviation usage and grammatical errors require careful revision throughout the\nmanuscript. For instance, the abbreviation for \"cryo-electron microscopy\" appears inconsistently as both\n\"cryoEM\" and \"cryo-EM\" (e.g., Lines 28 and 314, respectively). Please ensure consistent use of \"cryo-EM\"\nthroughout the text and introduce the full term before its first abbreviation.\n>>We have updated the text to define cryo-EM on first use and now use “cryo-EM” throughout\nthe text. All of the edits and inconsistencies below have been addressed and marked with an “x”\nbelow.\nHere are the comments presented as a point-wise list for the authors:\nx Abbreviations: Ensure consistency in the abbreviation \"cryo-EM\" throughout the text. Please use \"cryo-EM\"\ninstead of \"cryoEM\" (e.g., Line 28, 109, 111, 136). Always use the full term before introducing an abbreviation\nfor the first time.\nx Line 42: Remove the comma before \", cell attachment proteins\".\nx Line 46: Capitalize \"Gram-negative\".\nx Line 48: Remove the comma after \"rod-shaped,\".\nx Line 75: Change \"(mAb)\" to \"(mAbs)\".\nx Line 109: Add a full stop at the end of the sentence.\nx Line 113: The sentence \"Full fimbrial tips (FimH/G/F with chaperone FimC) complexed with multiple Fabs\nwithout overlapping epitopes increased the size of our target while allowing us to unambiguously identify each\nFab bound to FimH (Figure 1a-d, Ext. Data Fig. 5a).\" is lengthy and could be broken down for improved\nreadability.\nx Line 125: Add a hyphen to \"ligand-binding\".\nx Lines 323-331: Change \"beta strand\" to \"beta-strand\" and \"beta strands\" to \"beta-strands\".\n>>Note: we have updated all strand/sheet references to β-strands/sheets.\nx Line 506: Add a full stop at the end of the sentence.\nx Line 557: Change \"...plated on LB agar overnight.\" to \"...plated on LB agar and incubated overnight.\"\nx Line 559: Consider revising to: \"...induced by the addition of 0.5 mM IPTG, followed by overnight growth at\n22 °C.\"\nx Line 565: Change \"...size exclusion chromatography on a Superdex 75 column.\" to \"...size-exclusion\nchromatography using a Superdex 75 column.\"\nx Line 573: Change \"...size exclusion chromatography on a Superdex 200 column in PBS buffer.\" to \"...size-\nexclusion chromatography using a Superdex 200 column in PBS buffer.\"\nx Line 574: Change \"...digested to Fab fragments using agarose-immobilized ficin from Thermo Fisher\nScientific and purified by size exclusion on a Superdex 75 column.\" to \"...digested to Fab fragments using\nagarose-immobilized ficin (Thermo Fisher Scientific) and purified by size-exclusion chromatography using\na Superdex 75 column.\"\nx Line 578: Change \"...in a 1:1.2 ratio.\" to \"...at a 1:1.2 molar ratio.\"\nx Lines 596-597: Change \"100-300 Å min-max diameter\" to \"minimum and maximum particle diameters of 100-\n300 Å\".\nx Line 627: Change \"Glycopeptide sample processing:\" to \"Glycopeptide Sample Processing\".\nx Line 628: Change \"Each aliquot of individual Fabs contained 5 µg of the Fab molecule in 200 mM Tris buffer\nsolution...\" to \"Each aliquot of individual Fabs (5 µg) was prepared in 200 mM Tris buffer solution...\"\nx Line 645: Change \"Glycopeptide data acquisition:\" to \"Glycopeptide Data Acquisition\".\nx Line 668: Change \"PNGase F-treated peptide data acquisition:\" to \"PNGase F-treated Peptide Data\nacquisition:\".\n>>Note: we have changed the subsection title to “PNGase F-treated Peptide Data Acquisition:...”\nx Line 676: Change \"Glycopeptide data analysis:\" to \"Glycopeptide Data Analysis:\".\nx Line 677: Consider revising to: \"...with each search using the Fab-specific FASTA sequence file.\"\nx Lines 686 and 688: Change \"Oglycan\" to \"O-glycan\".\nx Line 712: Change \"Mab\" to \"mAbs\".\nx Line 714: Change \"microgram\" to \"µg\" and \"microliter\" to \"µL\".\nx Line 716: Change \"Reaction was performed at 37oC overnight.\" to \"Reaction was performed at 37 °C\novernight.\"\nx Line 718: Change \"Antiserum adsorption:\" to \"Antiserum Adsorption:\".\nx Line 725: Suggestion: \"Adsorption was performed by rotating the tubes at 16 rpm at room temperature for 1\nh.\"\nx Line 726: Change \"Upon the completion\" to \"Upon completion\", \".45\" to \"0.45\", and \"uM\" to \"µm\".\nx Line 729: Change \"Enzyme-linked immunosorbent assay:\" to \"Enzyme-linked Immunosorbent Assay:\".\nx Lines 736-737: Correct \" T-test and \"ttesti\" to the appropriate statistical test notation (e.g., t-test).\nx Line 740: Change \"Sequence Alignments of mAB475-like Antibodies:\" to \"Sequence Alignments of mAb475-\nlike Antibodies:\".\nx Line 746: Change \"V-Region\" to \"V-regions\" and \"manufacturer’s\" to \"the manufacturer's\".\nx Line 763: Change \"cryo EM\" to \"cryo-EM\".\nRESPONSE TO REVIEWERS' COMMENTS\n>>We thank the reviewers for their positive assessment of our revisions and address the minor comments\nbelow (>> and in italics).\nReviewer #1 (Remarks to the Author):\nThe authors have extensively revised their ms and perform several new simulations and experiments.\nOverall, they have satisfactorily addressed my comments. Unless I missed it, a version of the ms highlighting\nthe changes with color would have been appreciated to make the review process easier.\nAs a minor comment, I still believe that the presentation of the molecular mechanism of FimH binding is\ninaccurate or at best confusing. The current phrasing conveys the impression that ligand binding is the\nallosteric modulator, whereas it is mechanical force that effectively triggers ligand binding by stabilizing the\nhigh-affinity conformation).\n>>In our revision submission from the beginning of August, we included an MS word DOCX version of the\nmanuscript with changes tracked, where changes are indicated by underlining and text in color. This should\nhave been part of the packet available to reviewers; apologies for the confusion.\n>>We have updated the text to clarify that shear force is the allosteric modulator. Changes are tracked here\nwith underlined text.\n“FimH also serves as a model allosteric protein as it undergoes dramatic\nconformational rearrangements that decrease ligand dissociation7,10. In the\nabsence of ligand and tensile\nmechanical force, the FimH lectin domain adopts a compressed and inactive conformation\nstabilized by a fixed interface between the lectin and pilin domains, with the\nligand-binding pocket relaxed and open10 (Supp Fig. 1C). FimH can adopt an intermediate conformation,\nwhere a ligand occupies the binding site but no mechanical force is applied. In this\nconformation, the mannose-binding loops, N-terminus, and clamp loop encircle the ligand, but\nthe lectin-pilin domain interface remains fixed11 (Supp Fig. 1D). Ligand binding without\nmechanical force alters the equilibrium constant of lectin-pilin domain separation in FimH11–13.\nThis change increases the proportion of FimH that adopts an elongated and active conformation\nwhere the pilin and lectin domains are separated, although the domain-separated conformation\nof full length, ligand bound FimH is unstable and less populated in the absence of force (Supp\nFig. 1E). Allosteric modulation by tensile mechanical force causes lectin-pilin domain separation,\npulling the pilin domain away from the lectin domain and stabilizing conformational\nrearrangements and ligand binding in the lectin domain. The result is a force-enhanced catch-\nbond interaction between the ligand and the lectin domain7,10,14,15.”\nReviewer #2 (Remarks to the Author):\nThis re-submission has addressed in excellent detail all of the concerns and questions posed by the three\nreviewers.\nThe breadth and detail of their responses provide adequate clarifications of their work, and justify its\nimportance to the broad scientific community.\n>>Thank you.\nReviewer #3 (Remarks to the Author):\nThank you for the opportunity to review the revised manuscript \"NCOMMS-25-08562-T\". The authors have\nthoroughly addressed my concerns. Their revisions clarify several key points and significantly strengthen the\nmanuscript.\nThe authors have successfully addressed the discrepancy I noted between the cryo-EM data and the reported\nglycan analysis of Fab 475. They have updated the manuscript with an improved Ext. Data Fig. 3 and an\nadditional figure and note to clarify the differences between N28 and N90 glycans. The revised text and\nupdated methods section now clearly explain the glycoproteomics analysis workflow, making this section much\nmore robust.\nFor my second comment, the authors performed a crucial database search against the mouse proteome. This\nstep is crucial for eliminating false identifications, and they have incorporated this analysis into the revised\nmanuscript, addressing my concern regarding Ext. Data Fig. 8. The methods section has also been updated\nwith detailed criteria for the manual verification of glycopeptide identifications, which is a critical aspect of their\nanalysis. Regarding my fourth comment, the authors explained that they used a truncated version of Fab 926\nfor the molecular dynamics (MD) simulation to reduce computational cost. This detail has been added to the\nmethods section. For my fifth comment, they've clearly specified the rationale for the specific ratio of fimbrial\ntips to Fab fragments in the methods section. They also clarified the analysis workflow mentioned in my sixth\ncomment, detailing the use of two enzymes, trypsin and chymotrypsin, and providing the specifics in the\nmethods section. Additionally, the authors have addressed all other minor corrections.\nI am pleased that the authors have successfully addressed all my comments, particularly those regarding\nglycopeptide identification and the detailed parameters of their analyses.\nWith all of the modifications and revisions, I believe this article is now suitable for publication in Nature\nCommunications.\n>>Thank you."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have extensively revised their ms and perform several new simulations and experiments.\nOverall, they have satisfactorily addressed my comments. Unless I missed it, a version of the ms highlighting the changes\nwith color would have been appreciated to make the review process easier.\nAs a minor comment, I still believe that the presentation of the molecular mechanism of FimH binding is inaccurate or at best\nconfusing. The current phrasing conveys the impression that ligand binding is the allosteric modulator, whereas it is\nmechanical force that effectively triggers ligand binding by stabilizing the high-affinity conformation).", "reviewer_2": "This re-submission has addressed in excellent detail all of the concerns and questions posed by the three reviewers.\nThe breadth and detail of their responses provide adequate clarifications of their work, and justify its importance to the broad\nscientific communtiy.", "reviewer_3": "Thank you for the opportunity to review the revised manuscript \"NCOMMS-25-08562-T\". The authors have thoroughly\naddressed my concerns. Their revisions clarify several key points and significantly strengthen the manuscript.\nThe authors have successfully addressed the discrepancy I noted between the cryo-EM data and the reported glycan\nanalysis of Fab 475. They have updated the manuscript with an improved Ext. Data Fig. 3 and an additional figure and note\nto clarify the differences between N28 and N90 glycans. The revised text and updated methods section now clearly explain\nthe glycoproteomics analysis workflow, making this section much more robust.\nFor my second comment, the authors performed a crucial database search against the mouse proteome. This step is crucial\nfor eliminating false identifications, and they have incorporated this analysis into the revised manuscript, addressing my\nconcern regarding Ext. Data Fig. 8. The methods section has also been updated with detailed criteria for the manual\nverification of glycopeptide identifications, which is a critical aspect of their analysis.\nRegarding my fourth comment, the authors explained that they used a truncated version of Fab 926 for the molecular\ndynamics (MD) simulation to reduce computational cost. This detail has been added to the methods section. For my fifth\ncomment, they've clearly specified the rationale for the specific ratio of fimbrial tips to Fab fragments in the methods section.\nThey also clarified the analysis workflow mentioned in my sixth comment, detailing the use of two enzymes, trypsin and\nchymotrypsin, and providing the specifics in the methods section. Additionally, the authors have addressed all other minor\ncorrections.\nI am pleased that the authors have successfully addressed all my comments, particularly those regarding glycopeptide\nidentification and the detailed parameters of their analyses.\nWith all of the modifications and revisions, I believe this article is now suitable for publication in Nature Communications."}, "author_response": "RESPONSE TO REVIEWERS' COMMENTS\n>>We thank the reviewers for their positive assessment of our revisions and address the minor comments\nbelow (>> and in italics).\nReviewer #1 (Remarks to the Author):\nThe authors have extensively revised their ms and perform several new simulations and experiments.\nOverall, they have satisfactorily addressed my comments. Unless I missed it, a version of the ms highlighting\nthe changes with color would have been appreciated to make the review process easier.\nAs a minor comment, I still believe that the presentation of the molecular mechanism of FimH binding is\ninaccurate or at best confusing. The current phrasing conveys the impression that ligand binding is the\nallosteric modulator, whereas it is mechanical force that effectively triggers ligand binding by stabilizing the\nhigh-affinity conformation).\n>>In our revision submission from the beginning of August, we included an MS word DOCX version of the\nmanuscript with changes tracked, where changes are indicated by underlining and text in color. This should\nhave been part of the packet available to reviewers; apologies for the confusion.\n>>We have updated the text to clarify that shear force is the allosteric modulator. Changes are tracked here\nwith underlined text.\n“FimH also serves as a model allosteric protein as it undergoes dramatic\nconformational rearrangements that decrease ligand dissociation7,10. In the\nabsence of ligand and tensile\nmechanical force, the FimH lectin domain adopts a compressed and inactive conformation\nstabilized by a fixed interface between the lectin and pilin domains, with the\nligand-binding pocket relaxed and open10 (Supp Fig. 1C). FimH can adopt an intermediate conformation,\nwhere a ligand occupies the binding site but no mechanical force is applied. In this\nconformation, the mannose-binding loops, N-terminus, and clamp loop encircle the ligand, but\nthe lectin-pilin domain interface remains fixed11 (Supp Fig. 1D). Ligand binding without\nmechanical force alters the equilibrium constant of lectin-pilin domain separation in FimH11–13.\nThis change increases the proportion of FimH that adopts an elongated and active conformation\nwhere the pilin and lectin domains are separated, although the domain-separated conformation\nof full length, ligand bound FimH is unstable and less populated in the absence of force (Supp\nFig. 1E). Allosteric modulation by tensile mechanical force causes lectin-pilin domain separation,\npulling the pilin domain away from the lectin domain and stabilizing conformational\nrearrangements and ligand binding in the lectin domain. The result is a force-enhanced catch-\nbond interaction between the ligand and the lectin domain7,10,14,15.”\nReviewer #2 (Remarks to the Author):\nThis re-submission has addressed in excellent detail all of the concerns and questions posed by the three\nreviewers.\nThe breadth and detail of their responses provide adequate clarifications of their work, and justify its\nimportance to the broad scientific community.\n>>Thank you.\nReviewer #3 (Remarks to the Author):\nThank you for the opportunity to review the revised manuscript \"NCOMMS-25-08562-T\". The authors have\nthoroughly addressed my concerns. Their revisions clarify several key points and significantly strengthen the\nmanuscript.\nThe authors have successfully addressed the discrepancy I noted between the cryo-EM data and the reported\nglycan analysis of Fab 475. They have updated the manuscript with an improved Ext. Data Fig. 3 and an\nadditional figure and note to clarify the differences between N28 and N90 glycans. The revised text and\nupdated methods section now clearly explain the glycoproteomics analysis workflow, making this section much\nmore robust.\nFor my second comment, the authors performed a crucial database search against the mouse proteome. This\nstep is crucial for eliminating false identifications, and they have incorporated this analysis into the revised\nmanuscript, addressing my concern regarding Ext. Data Fig. 8. The methods section has also been updated\nwith detailed criteria for the manual verification of glycopeptide identifications, which is a critical aspect of their\nanalysis. Regarding my fourth comment, the authors explained that they used a truncated version of Fab 926\nfor the molecular dynamics (MD) simulation to reduce computational cost. This detail has been added to the\nmethods section. For my fifth comment, they've clearly specified the rationale for the specific ratio of fimbrial\ntips to Fab fragments in the methods section. They also clarified the analysis workflow mentioned in my sixth\ncomment, detailing the use of two enzymes, trypsin and chymotrypsin, and providing the specifics in the\nmethods section. Additionally, the authors have addressed all other minor corrections.\nI am pleased that the authors have successfully addressed all my comments, particularly those regarding\nglycopeptide identification and the detailed parameters of their analyses.\nWith all of the modifications and revisions, I believe this article is now suitable for publication in Nature\nCommunications.\n>>Thank you."}]

Decision letter