Inhibition of Mammalian Glycoprotein YKL-40 IDENTIFICATION OF THE PHYSIOLOGICAL LIGAND

14 Pages·2017·2.39 MB·English

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THEJOURNALOFBIOLOGICALCHEMISTRY VOL.292,NO.7,pp.2624–2636,February17,2017
©2017byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Inhibition of Mammalian Glycoprotein YKL-40
IDENTIFICATIONOFTHEPHYSIOLOGICALLIGAND*□S
Receivedforpublication,October26,2016,andinrevisedform,December22,2016 Published,JBCPapersinPress,January4,2017,DOI10.1074/jbc.M116.764985
AbhishekA.KognoleandX ChristinaM.Payne1
FromtheDepartmentofChemicalandMaterialsEngineering,UniversityofKentucky,Lexington,Kentucky40506
EditedbyGeraldW.Hart
YKL-40 is a mammalian glycoprotein associated with pro- easesandamultitudeofcancers(1–4).Manydifferenttypesof
gression,severity,andprognosisofchronicinflammatorydis- cellsincludingsynovial,endothelial,epithelial,smoothmuscle,
easesandamultitudeofcancers.Despitethiswelldocumented andtumorcellsproduceYKL-40invivo,likelyinresponseto
association, identification of the lectin(cid:3)s physiological ligand environmentalcues(5–8).Speculationastobiologicalfunction
and,accordingly,biologicalfunctionhasprovenexperimentally ofYKL-40variesfrombothinhibitingandantagonizingcolla-
difficult. YKL-40 has been shown to bind chito-oligosaccha- genfibrilformationasaresultofinjuryordisease(9),aswellas
rides;however,theproductionofchitinbythehumanbodyhas conferringdrugresistanceandincreasingcellmigrationlead-
notyetbeendocumented.Possiblealternativeligandsinclude ing to progression of cancer (3), and protection from chitin-
proteoglycans, polysaccharides, and fibers like collagen, all of containingpathogens(10).AlthoughtheassociationofYKL-40
whichmakeuptheextracellularmatrix.ItislikelythatYKL-40is withphysicalmaladiesiswelldocumented,identificationofthe
interacting with these alternative polysaccharides or proteins
physiologicalligandofthislectin,andthusbiologicalfunction,
withinthebody,extendingitsfunctiontocellbiologicalroles
remainselusive.
such as mediating cellular receptors and cell adhesion and
Mammalian YKL-40 is classified as a family 18 glycoside
migration.Here,weconsiderthefeasibilityofpolysaccharides,
hydrolase based on high homology with this well conserved
including cello-oligosaccharides, hyaluronan, heparan sulfate,
classofenzymesintheCAZydatabase(10–12).Althoughsim-
heparin,andchondroitinsulfate,andcollagen-likepeptidesas
ilarinstructuretofamily18glycosidehydrolases,YKL-40lacks
physiologicalligandsforYKL-40.Weusemoleculardynamics
catalyticactivityasaresultofsubstitutionoftheglutamicacid
simulationstoresolvethemolecularlevelrecognitionmecha-
andasparticacidmotiftypicalofcatalyticallyactivefamily18
nismsandcalculatethefreeenergyofbindingthehypothesized
hydrolases, rendering YKL-40 a lectin, a non-catalytic sugar-
ligandstoYKL-40,addressingthermodynamicpreferencerela-
binding protein. Structural evidence suggests that YKL-40
tive to chito-oligosaccharides. Our results suggest that chito-
exhibitsatleasttwofunctionalbindingregions(10).Thepri-
hexaoseandhyaluronanpreferentiallybindtoYKL-40overcol-
mary binding cleft has nine binding subsites lined with aro-
lagen, and hyaluronan is likely the preferred physiological
maticresiduescompatiblewithcarbohydratebinding(Fig.1).A
ligand, because the negatively charged hyaluronan shows
putativeheparin-bindingsite,locatedwithinasurfaceloop,has
enhancedaffinityforYKL-40overneutralchitohexaose.Colla-
alsobeensuggested(Fig.1),althoughinvitrobindingaffinity
gen binds in two locations at the YKL-40 surface, potentially
studieshavebeenunabletoconclusivelydemonstratethis(11).
relatedtoaroleinfibrillarformation.Finally,heparinnon-spe-
Binding affinity and structural studies reveal that chito-
cificallybindsattheYKL-40surface,aspredictedfromstruc-
oligosaccharidesarenaturalsubstrates(6,10,11).Inlinewith
turalstudies.Overall,YKL-40likelybindsmanynaturalligands
family 18 glycoside hydrolases, YKL-40 uniquely binds short
in vivo, but its concurrence with physical maladies may be
andlongchito-oligomers,indicatingpreferentialsiteselection
relatedtoassociatedincreasesinhyaluronan.
basedonaffinity(10).Chitohexaosebindinghasalsobeenpur-
ported to induce conformational changes in YKL-40 (10),
although this has not been observed in all structural studies
YKL-40, also known as chitinase 3-like 1, is a mammalian
(11). Lectin binding niches are widely believed to be “pre-
glycoprotein implicated as a biomarker associated with pro-
formed” to the preferred ligand, exhibiting little conforma-
gression,severity,andprognosisofchronicinflammatorydis-
tionalchangeuponbinding(13,14).Despiteapparentaffinity,
chitinisnotanaturalbiopolymerwithinmammalianorbacte-
*ThisworkwassupportedbyKentuckyScienceandEngineeringFoundation rialcells,andthepresenceofchitinorchito-oligosaccharidesin
Grant KSEF-148-502-13-307). Computational time for this research was
mammals is likely related to fungal infection (15). The noted
providedbytheExtremeScienceandEngineeringDiscoveryEnvironment
(83), which is supported by National Science Foundation Grant ACI- up-regulation of YKL-40 in response to inflammation lends
1053575underAllocationMCB090159.Theauthorsdeclarethattheyhave credencetotheargumentthatYKL-40functionsaspartofthe
noconflictsofinterestwiththecontentsofthisarticle.
□S This article contains supplemental text, Tables S1–S6, Figs. S1–S8, and innateimmuneresponseinrecognitionofselffromnon-self(6,
MoviesS1–S3. 16); however, high expression levels of YKL-40 in carcinoma
1To whom correspondence should be addressed: Dept. of Chemical and tissues suggest function beyond the innate immune response
MaterialsEngineering,UniversityofKentucky,177F.PaulAndersonTower,
mayalsoexist(17,18).Theextracellularmatrixiscomprisedof
Lexington, KY 40506. Tel.: [phone]; Fax: [phone]; E-mail:
[email protected]. a mesh of proteoglycans (protein-attached glycosaminogly-
This is an Open Access article under the CC BY license.
2624 JOURNALOFBIOLOGICALCHEMISTRY VOLUME292•NUMBER7•FEBRUARY17,2017
IdentifyingthePhysiologicalLigandofYKL-40
therconfoundsthequestionofmechanismwhenconsidering
physiological ligands, because YKL-40 is capable of binding
bothproteinandcarbohydrates.
UnderstandingthemechanismandaffinitybywhichYKL-40
bindsligandsiscrucialtoourunderstandingofitsphysiological
function.Thisknowledgewillserveasafoundationforfuture
campaigns toward rational development of a potent antago-
nistsenablingcellbiologicalstudyandaddressingYKL-40asa
therapeutictarget.Toaccomplishthisgoal,wemustdescribe
the molecular level mechanisms governing the interaction of
YKL-40 with both polysaccharide and collagen-like polypep-
tidesandquantitativelyevaluateaffinity.Inthisstudy,weused
FIGURE1.SurfacerepresentationofYKL-40showingthebindingcleft classicalmoleculardynamics(MD)2simulationstodifferenti-
withaboundhexamerofchitin.Bindingsites(cid:2)2through(cid:3)4arenum-
ate modes of ligand recognition and specificity. Using free
bered.Sites(cid:3)5,(cid:3)6,and(cid:3)7havealsobeenidentifiedbutarenotshown.The
putativeheparin-bindingsiteisshowninblue. energyperturbationwithreplicaexchangemoleculardynamics
(FEP/(cid:2)-REMD)andumbrellasamplingMD,wequantitatively
cans),polysaccharides,andfibersincludingcollagen.Analter- determinedaffinitiesovercomingtheexperimentaldifficulties
nate theory to the pathogenic protection function is that a encounteredthusfar.Asphysiologicalligands,weconsidered
closelyrelatedpolysaccharide,insteadofchitin,playstheroleof several options within both the polysaccharide and proteina-
thephysiologicalligandinmediatingcellularfunction(11). ceous classes. Below, we provide a brief description of each
Despitethestructuralsimilaritybetweenchito-oligosaccha- carbohydrateligandconsidered,aswellasjustificationforcon-
ridesandtheproteoglycancarbohydratemonomers,littleevi-
siderationofthecollagen-likemodels.
denceofpolysaccharidebindingbeyondtheoriginalstructural
Polysaccharides—Weselectedpolysaccharidesforthisstudy
studiesexists(10,11).Infact,weareawareofonlyoneother
basedontheirsimilaritytochito-oligomers,aswellasnatural
studyfocusingonthemolecular-levelmechanismofcarbohy-
occurrence in mammalian cell walls and/or the extracellular
dratebindinginYKL-40(19).Fromabioinformaticsandstruc-
matrix. The chito-oligomer from structural studies was
turalcomparisonofYKL-40toasimilarchi-lectin,mammary
included as a control (10, 11). Chitin is a naturally occurring
glandprotein40(20),theauthorsproposeanoligosaccharide
biopolymer comprised of repeating N-acetyl-D-glucosamine
bindingmechanismthatinvolvestryptophan-mediatedgating (GlcNAc)monomericunitsconnectedby(cid:3)-1,4-glycosidiclink-
oftheprimarycarbohydratebindingsite(Fig.1)(19).However,
ages(Fig.2).Thecentralringofthemonomer,asix-membered
in lieu of a dynamics-based investigation, little can be con-
pyranose,iscommontoanumberofcarbohydratesincluding
cludedaboutthebindingmechanismofYKL-40ligandsother
glucose. Given the chemical similarity, as well as the general
thanchito-oligosaccharides,andconformationalchangesrela-
presenceofglucoseinmammaliancellsasaformofenergy,a
tivetobindingareinaccessible.Fromproteinpurificationtech-
hexameric cello-oligomer was also examined as a potential
niques, namely heparin-Sepharose chromatography, we also
physiologicalligand,despiteitsunlikelypresenceamongmam-
knowthatYKL-40reversiblybindsheparin(7,11,21);however,
malianglycosaminoglycans.
affinitydataforthisinteractiondonotexist.Basedontheinter-
Asdescribedabove,YKL-40bindsheparin,andthus,likely
action with heparin, it is reasonable to hypothesize heparan
alsobindsheparansulfate.Heparansulfate,alesssulfatedform
sulfateglycosaminoglycans,existingaspartoftheextracellular
ofheparin,isapolysaccharidefoundinabundanceintheextra-
matrix construct, are potential physiological ligands. Visual
cellularmatrixandatthecellsurface(27).Heparansulfateis
inspectionoftheproteinstructuresuggeststhatheparansul-
constructed from a repeating disaccharide of (cid:3)-D-glucuronic
fatefragmentsmaybeeasiertoaccommodatewithinthecar-
acidandN-acetyl-(cid:4)-D-glucosamine(Fig.2).Ofalltheglycos-
bohydrate-bindingsitethanheparinitself(11).Itfollowsthat
aminoglycans,heparansulfateisthemoststructurallycomplex.
otherstructurallysimilarcarbohydratefragmentswouldbind
Atleast24differentcombinationsofthedisaccharidemono-
withsimilaraffinityinacomparablemechanism.
merexist,withdifferencesarisingasaresultofvariationinboth
The association of YKL-40 with ailments such as arthritis,
isomeranddegreeofsidechainsulfation(28).Additionally,the
fibrosis,andjointdiseaseissuggestiveofmolecular-levelinter-
heparan sulfate polysaccharide can exhibit both sulfated and
actionswithconnectivetissueandthuscollagen(22–26).Moti-
unsulfated domains. Physiologically, the unsulfated disaccha-
vated by understanding the physiological role of YKL-40 in
connectivetissueremodelingandinflammation,Biggetal.(9) ride(cid:3)-D-glucuronicacid(1,4)N-acetyl-(cid:4)-D-glucosamineisthe
most prevalent form of heparan sulfate (28). Focusing on the
investigatedassociationofYKL-40withcollagentypesI,II,and
mostrelevantphysiologicalligands,weexaminedthefullysul-
III using affinity chromatography to confirm binding to each
fatedformheparinandtheunsulfatedformheparansulfate.
type.TheauthorsreportYKL-40specificallybindstoallthree
collagentypes.Additionally,theauthorsusedsurfaceplasmon
resonancetoconfirmbindingtocollagentypeI.Unfortunately,
thereportedaffinityconstantswereinconsistentacrossexper- 2The abbreviations used are: MD, molecular dynamics; PMF, potential of
meanforce;FEP,freeenergyperturbation;REMD,replicaexchangemolec-
imentsasaresultofaggregation.Nevertheless,theworkclearly
ulardynamics;WCA,Weeks-Chandler-Anderson;RMSD,rootmeansquare
indicatesthatYKL-40iscapableofbindingcollagen.Thisfur- deviation;RMSF,rootmeansquarefluctuation;PDB,ProteinDataBank.
FEBRUARY17,2017•VOLUME292•NUMBER7 JOURNALOFBIOLOGICALCHEMISTRY 2625
IdentifyingthePhysiologicalLigandofYKL-40
Gly-Xaa-Yaa repeating amino acid sequence (34). Generally,
the unspecified amino acids, Xaa and Yaa, are proline and
hydroxyproline,respectively.
Modelcollagenpeptideshavebeenobservedintwodifferent
symmetries:theoriginalRichandCrickmodelwith10/3sym-
metry (10 units in 3 turns) and the 7/2 symmetry of a more
tightly symmetrical triple helix (35–37). On the molecular
scale,collagentypewillhaverelativelylittleimpactonbinding
toYKL-40.However,symmetrymayhaveanimpactonhydro-
genbondinginthebindingsite,andthus,overallaffinity,which
will provide unique insight into physiological relevance. To
date,modelcollagenpeptidesofatrue10/3symmetryhavenot
been reported. Rather, the peptides either have a 7/2 helical
pitch or are somewhat “intermediate” in symmetry leading
sometobelievethatthe7/2symmetryisrepresentativeofthe
true collagen helical structure (38). However, it is not known
howuniversallytruethishypothesisisbecausethestructuresof
model peptides capture just a small subsection of the larger
macromolecularstructure(39).
With a broad range of possible collagen architectures, we
have selected four representative model collagen peptides
FIGURE2.Monomericunitsofthepolysaccharidesconsideredaspoten- whosestructuresarebothavailablefromcrystallographicevi-
tialphysiologicalligandsofYKL-40:cellohexaose,chitohexaose,hepa-
dence and span the 10/3 and 7/2 symmetries to the greatest
ransulfate,heparin,hyaluronan,andchondroitinsulfate.Thechito-oli-
gomerisapolymerof(cid:3)-1,4-linkedGlcNAcmonomers.Heparansulfatewas possibleextent.Thefirstcollagenpeptideconsideredisthatof
modeledasa(cid:3)-1,4,(cid:4)-1,4-linkedchainofGlcAandGlcNAc.Heparinwasrep-
the basic collagen peptide model, PDB code 1CAG (40). The
resentedasthe(cid:3)-1,4,(cid:4)-1,4-linkedoligomerofGlcAandGlcNS.Hyaluronan
andchondroitinsulfateare(cid:3)-1,3,(cid:3)-1,4-linkedoligomers;theformerconsists reported1.9ÅresolutionstructureexhibitsasingleGlytoAla
ofGlcAandGlcNAc,andthelatterconsistsofGlcAandGalNAc.GlcA,(cid:3)-D- substitution and 7/2 symmetry overall. Near the substitution
glucuronicacid;IdoA,(cid:4)-D-iduronicacid;GlcNS,N-sulfo-(cid:4)-D-glucosamine;Gal-
NAc,N-acetyl-(cid:3)-D-galactosamine. site,thehelixrelaxessomewhatfrom7/2symmetry,although
notsomuchastochangeoverallsymmetry.Thesecondcolla-
genmodelpeptideweconsiderisavariationofthe1CAGpep-
Hyaluronan is a particularly interesting glycosaminoglycan tide, where we reverted the alanine substitution to its native
relative to this study, because chito-oligosaccharides are pre- glycine.Minimizationofthisstructurereturnsthehelixtofull
cursorstohyaluronansynthesisinvivo(29–31).Thestructural 7/2 symmetry; we refer to this peptide as “native 1CAG”
relationshipofthesetwomoleculesissuchthatbindingmech- here. The third model represents a segment from type III
anismsmaybesimilaratalternatingbindingsites.Hyaluronan homotrimercollagenwithapproximate10/3symmetryinthe
is a polysaccharide of a repeating (cid:3)-D-glucuronic acid and middle part of the helix (PDB code 1BKV) (35). This middle
N-acetyl-(cid:3)-D-glucosamine disaccharides connected by alter- part of the 1BKV model peptide, also referred as the T3–785
nating (cid:3)-1,3- and (cid:3)-1,4-glycosidic linkages (Fig. 2). As with
peptide, has an imino acid-poor sequence of GITGARGLA.
heparansulfate,hyaluronanisalsoaglycosaminoglycancom-
Ourfourthmodel,1Q7D,isatriplehelicalcollagen-likepeptide
prising the extracellular matrix. At extracellular pH, the car-
sequence including a hexapeptide Gly-Phe-Hyp-Gly-Glu-Arg
boxyl groups of glucuronic acid are fully ionized, giving the
(GFOGER) motif in the middle (41); this motif is not suffi-
ligand an overall negative charge under typical physiological
ciently long to exhibit 10/3 symmetry, exhibiting, rather, an
conditions(32).
intermediatedegreeof7/2helicalsymmetry.This1Q7Dmodel
Finally,weconsiderchondroitinsulfate,whichisalsoagly- isknowntobindtheintegrin(cid:4)2(cid:3)1-Idomainprotein(42),and
cosaminoglycanprevalentinmammals.Theprimarystructural theGFOGERmotifisfoundinthe(cid:4)1chainoftypeIcollagen.
unitsofchondroitinsulfatearearepeating(cid:3)-D-glucuronicacid
and N-acetyl-(cid:4)-D-galactosamine disaccharides connected by ExperimentalProcedures
alternating(cid:3)-1,3-and(cid:3)-1,4-glycosidiclinkages(Fig.2).Aswith
MolecularDynamicsSimulations
heparansulfate,chondroitinsulfateexistsinvariablysulfated
types(33);wehaveselectedtheC4andC6sulfatedvariantof MD simulations were constructed starting from the chito-
chondroitinsulfatepolysaccharideasamodel. hexaose-boundYKL-40structure(PDBcode1HJW)deposited
Collagen-like Peptide Models—Collagen has also been con- byHoustonetal.(10).Theaposimulationsimplyremovedthe
sideredasapotentialphysiologicalligandbasedonthenoted chito-oligomer from the primary binding cleft. The N-linked
affinityandparticipationofYKL-40incollagenfibrilformation glycancapturedintheYKL-40structure,atAsn60,wasincluded
(9).Collagen,unliketheotherpotentialphysiologicalligands,is in system preparation. Because crystal structures of YKL-40
amacromolecularproteinwithatriplehelixstructure.There boundtootherpolysaccharidesarenotavailable,weusedthe
areatleast27distincttypesofhumancollagen,formingavari- structuralsimilarityofpolysaccharidesasthebasisformodel-
etyofbiologicalnetworks,allofwhichareconstructedofabasic ingtheremainingpolysaccharidesinthisinvestigation.Inthe
2626 JOURNALOFBIOLOGICALCHEMISTRY VOLUME292•NUMBER7•FEBRUARY17,2017
IdentifyingthePhysiologicalLigandofYKL-40
FIGURE3.Molecularshapecomplementaritydockingcalculationspredictcollagen-likepeptideswillbindtoYKL-40intwopossibleorientations.a,
thefrontviewofYKL-40(graysurface)withcollagendockedinsiteA(greenstick).b,thebackviewwherecollagenisdockedinsiteB(cyanstick).c,topviewof
YKL-40illustratingrelativepositionsofbindingsites.Theputativeheparin-bindingsubsiteisshowninbluesurfacetoaidinvisualizationofrelativeorientation
oftheprotein(cid:4)proteincomplexes.Thisparticularfigureshowstheintegrin-bindingcollagenpeptide,1Q7D(41),inthepredictedbindingsitesalongthesurface
ofYKL-40;similardockingwascarriedoutforothercollagenmodels.
caseofcellohexaose,hyaluronan,heparansulfate,heparin,and 300K using NAMD (51). Explicit procedural details are pro-
chondroitin sulfate, we located the central ring atoms of the videdinthesupplementalmaterials.
ligand backbone in the same location as that of the original
chitohexaose.Appropriatepyranosesidechainsandglycosidic FreeEnergyCalculations
linkages(Fig.2)wereaddedusingCHARMMinternalcoordi- FEP/(cid:2)-REMD—FreeenergyperturbationwithHamiltonian
natetablestoconstructtheremainderofthesugarresidue(43). replicaexchangemoleculardynamics(FEP/(cid:2)-REMD)wasused
AllpolysaccharidesweredescribedusingtheCHARMM36car- tocalculatetheabsolutefreeenergyofbindingthepolysac-
bohydrateforcefield(44–46).Themissingforce-fieldparam- charideligandstoYKL-40(52,53).ThisprotocolusesHam-
etersforN-sulfatedglucosamine(supplementalFig.S1)inhep- iltonianreplicaexchangeasameansofimprovingtheBoltz-
arin were developed using the force-field Toolkit plugin for mannsamplingoffreeenergyperturbationcalculations.The
VMD (47, 48). Details of this parameterization and output parallel/parallel replica exchange MD algorithm in NAMD
parameters(supplementalTableS2)arereportedinthesupple- was implemented as recently described (51, 54). The free
mentalmaterials. energy calculations performed using this approach were
Construction of the collagen-bound YKL-40 models re- accomplished through two separate sets of free energy cal-
quired docking calculations to appropriately position the culations following the thermodynamic cycle illustrated in
ligand.Thecollagenpeptidesaresignificantlylargerthananyof supplemental Fig. S2. To obtain each binding free energy,
thecarbohydrateligands;thus,itisunlikelythatacollagenmol- (cid:4)G,theboundcarbohydrateligandwasfirstdecoupledfrom
ecule occupies the primary YKL-40 binding site in the same thesolvatedprotein(cid:4)carbohydratecomplextodetermine(cid:4)G .
1
manneraschito-oligomer.Standardaffinity-baseddockingcal- Thesecondcalculationentaileddecouplingthesolvatedoligo-
culations, such as the ones performed in AutoDock, are not saccharidefromsolutionintovacuumtoobtain(cid:4)G .Thedif-
2
feasiblefordeterminationofaninitialcollagen-bindingdomain ferencebetweenthetwovalues,(cid:4)G (cid:3)(cid:4)G ,givestheabsolute
2 1
given the size and flexibility of the triple helix structures. freeenergyofbindingthegivenligandtoYKL-40.
Rather, the collagen peptides were docked on the basis of Ineachfreeenergycalculation,fiveseparatetermscontrib-
molecularshapecomplementarityusingtheonlinewebserver utetothepotentialenergyofthesystem:thenon-interacting
PatchDockBetaversion1.3(49,50).Inthecaseofeachofthe ligandpotentialenergy,repulsiveanddispersivecontributions
four collagen-like model peptides, PatchDock predicted two totheLennard-Jonespotential,electrostaticcontributions,and
potentialoccupanciesalongthesurfaceofYKL-40,siteAand the restraining potential. In each calculation, the ligand was
siteB.BindingsiteAcorrespondstotheprimarycarbohydrate- decoupled from the system by thermodynamic coupling
binding domain of YKL-40; however, the collagen ligand was parameters controlling the non-bonded interaction of the
notasdeeplyentrenchedinthecleftaschitohexaose.Binding ligand with the environment. The parameters decoupled the
siteBislocatedontheoppositesideofYKL-40fromthepri- ligandinafour-stageprocess,whereinthecouplingparameters
mary binding cleft. Thus, for each collagen-like peptide, two defined replicas that were exchanged along the length of the
MDsimulationswereconstructedrepresentingthetwopoten- alchemical pathway. This decoupling has been described in
tialbindingsites.Fig.3illustratestheresultsofthedockingwith detailpreviously(54)andisalsoreportedinthesupplemental
predictedcollagenbindingsitesAandBforthe1Q7Dcollagen- materials.Atotalof128FEPreplicaswereused,andaconven-
likemodelpeptide. tional Metropolis Monte Carlo exchange criterion governed
Theconstructedprotein-ligandsystemswereminimizedin the swaps throughout the replica exchange process (53). The
vacuumandsubsequentlysolvatedwithwaterandsodiumions. free energy of binding was determined from 20 consecutive,
Using CHARMM (43), the solvated systems were extensively 0.1-ns simulations of each corresponding system, where the
minimizedandheatedto300Kfor20ps,whichwasfollowedby first1nsofdatawerediscardedasequilibration.Onestandard
MD simulation for 100 ps in the NPT ensemble. The coordi- deviationofthelast1nsofsimulationdatawereusedtoobtain
nates following density equilibration were used as a starting anestimateoferror.Additionaldetailsaboutthesecalculations
point for 250 ns of MD simulation in the NVT ensemble at aredescribedinthesupplementalmaterials.
FEBRUARY17,2017•VOLUME292•NUMBER7 JOURNALOFBIOLOGICALCHEMISTRY 2627
IdentifyingthePhysiologicalLigandofYKL-40
FIGURE4.RelaxationofthepolysaccharideligandsintheprimarybindingcleftofYKL-40.Eachligandisshownaftera100-psequilibrationinathickstick
representation.Forcomparison,thechito-oligomer,initsequilibratedconformation,isshowninthingreenlinesbehindeacholigosaccharide.TheYKL-40
proteinhasbeenalignedsuchthateacholigosaccharideisorientedinthesamemanner;althoughYKL-40isnotshownforvisualclarity.Heparansulfate,
heparin,andchondroitinsulfaterelaxsignificantlyfromtheinitialdistortedconformation.
UmbrellaSampling—ConvergencechallengesmakeFEP/(cid:2)- onlythreebindinastablefashionintheprimarycarbohydrate
REMDinappropriatefordeterminingthebindingfreeenergy binding site of YKL-40. The three potential polysaccharide
ofthemuchlargerandmoreflexiblecollagen-likemodelpep- physiological ligands at this site include chitohexaose, cello-
tides.Thus,umbrellasamplingwasusedtodeterminethework hexaose, and hyaluronan. In the section that follows, we will
requiredtodetachthecollagenligandsfromtheshallowclefts describethedynamicsofchitohexaose,cellohexaose,andhya-
of YKL-40. Over the entire reaction coordinate, this value luronan binding to YKL-40. The remaining three ligands—
equatestobindingaffinity,enablingrelativecomparisonofcol- heparin, heparan sulfate, and chondroitin sulfate—were dis-
lagenpeptideaffinity.TheMDumbrellasamplingsimulations lodgedfromthebindingsiteoverthecourseofMDsimulations.
usedanativecontacts-basedreactioncoordinateanalogousto The (cid:4)-1,4-glycosidic linkages in heparin and heparan sulfate,
that defined by Sheinerman and Brooks (55) and as imple- insteadof(cid:3)-1,4,modifiestherelativeorientationofdisaccha-
mented in recent cellulose decrystallization studies (56, 57). ride monomers from that of the chito-oligosaccharide. The
Here,anativecontactwasdefinedasaYKL-40proteinresidue
NMR solution structure of heparin (PDB code 1HPN) shows
within12Åofacollagenpeptideresidue;distancewasdefined
thattherelaxedconformationissemi-helical(59),whichcan-
by center of geometry of a given residue. The cutoff distance
notbefeasiblyaccommodatedintheconserved,narrowcarbo-
wasselectedtobelargerthanthenon-bondedcutoffdistance,
hydrate binding site of YKL-40. Heparan sulfate suffers from
ensuringthatthecollagenligandwasnolongerinteractingwith
similarstericconstraintsposedbytherelaxationdrivingforce.
YKL-40.Additionally,thewaterboxesofthecollagen-YKL-40
The bulky sulfated side chains of heparin introduce further
systemsweremadebiggertoaccommodatetherequiredsepa-
steric hindrance and, in the case of heparin and chondroitin
rationdistance.
sulfate, unfavorably strong electrostatic interactions resulting
Thechangeinfreeenergywasdeterminedasafunctionof
fromnegativelychargedmoietiesinconvenientlylocatedalong
thereactioncoordinate,(cid:5),formulatedastheweightedsumof
thecleft(i.e.withoutaco-located,oppositelychargedprotein
thestatesofthenativecontacts.Theinitialcoordinatesofthe
residue)ejecttheligandsfromthecleft.
bound systems were selected from 250-ns equilibrated snap-
Inthecasesofheparin,heparansulfate,andchondroitinsul-
shots.Theinitialnumberofnativecontactsandtheirweights
fate,theligandsquickly“relax”fromtheinitialwideV-shaped
werecalculatedfromthesesnapshots.Aninitialreactioncoor-
conformationastheyareexpelledfromthecleftbycharge-and
dinateof0(normalized)correspondstothisinitialcondition,
steric-based effects. Relaxation of the sugar from the initial
and a final reaction coordinate of 1 corresponds to all of the
bindingposeissufficienttoinitiatelossofcriticalnon-bonded
nativecontactsbeingoutsidethe12-Åcutoff(i.e.theligandis
interactionsalongwithasubsequentreductioninaffinity(Fig.
decoupledandfreelysamplingthebulk).Thereactioncoordi-
4).Within25ns,heparin,heparansulfate,andchondroitinsul-
natewasdividedinto20windowsevenlyspacedalongthereac-
fatewereexpelledfromthecleftintobulksolution.Eachofthe
tioncoordinate,andeachwindowwassampledfor5ns,where
threeligandscapableofbindingwiththeprimarybindingcleft
thereactioncoordinatewasmaintainedatthespecifiedvalue
maintainedthe(cid:3)1boatconformationovertheentiresimula-
usingaharmonicbiasingforcewiththeforceconstantof41,840
kJ/mol. The potentials of mean force profiles were calculated tion.Chitohexaoseandcellohexaoseremainedinthebinding
usingtheweightedhistogramanalysismethod(WHAMsoft- cleftovertheentire250-nsMDsimulationwhilemaintaining
ware),anderroranalysiswasperformedusingbootstrapping. theinitialwideVshape.HyaluronandevelopedasharpVshape
Adetailedlistingofallthesimulationsandfreeenergycalcula- within a few nanoseconds and maintained this conformation
tions performed as part of the objectives of this study is pro- within the binding cleft for the remainder of the simulation
videdinsupplementalTableS1. (supplementalFig.S6);thisisprimarilyduetovariationingly-
cosidic linkage, where hyaluronan exhibits a (cid:3)-1,3-linkage
ResultsandDiscussion withinthemonomerinsteadofthe(cid:3)-1,4-linkageofcello-and
Protein-PolysaccharideBindinginYKL-40—MDsimulation chitohexaose. Also, comparison of the equilibrated chito-
suggeststhatofthesixpolysaccharideoligomersinvestigated, hexaose- and hyaluronan-bound structures disabuses one of
2628 JOURNALOFBIOLOGICALCHEMISTRY VOLUME292•NUMBER7•FEBRUARY17,2017
IdentifyingthePhysiologicalLigandofYKL-40
FIGURE5.SnapshotsfromfourindependentMDsimulationsofheparin(whitestick)bindingtoaputativeheparin-bindingsite(bluesurface)ofYKL-40
(graysurface).TheprimaryoligosaccharidebindingsiteofYKL-40ismarkedbyanaromaticresidueshowninsalmonsurfacerepresentation.Transparent
spheresillustratetheinitialsimulationpositionsofheparin.Intwocases,aandb,theheparinligandwasinitiallyboundintheprimaryYKL-40bindingsite.In
bothcases,theligandwasexpelledfromtheprimarybindingsiteintosolutionandlocatedtheheparin-bindingsitethroughelectrostaticinteractions.Two
additionalsimulations,candd,wereinitializedwiththeheparinligandfreeinsolution.Again,theligandsidentifiedtheYKL-40heparin-bindingdomain
throughcharge-basedinteractions.Theliganddidnotspecificallybindinaparticularconformation.Rather,theliganddynamicallyinteractedwiththe
heparin-bindingdomain.
thenotionthatsimilarbindingmechanismsexistatalternate dentMDsimulationsoftheYKL-40/heparinsystem:onewitha
binding sites, because only the (cid:3)1 site pyranose appears to newrandomnumberseed,althoughinthesameconfiguration,
maintainsimilarsidechainorientation. andtwoadditionalsimulationswiththeligandrandomlyplaced
Thenativedistortedconformationischaracteristicofglyco- insolution(supplementalMovieS2).Ineachcase,theheparin
sidehydrolasepyranosebindingbehaviorinthe(cid:3)1site(Fig.4) oligomers were capable of finding and binding to a group
(60). In solution, polysaccharide pyranose moieties adopt the of charged residues at the surface of YKL-40 (Fig. 5); these
energeticallyfavorablechairconformation(61);however,when were the basic residues of a putative heparin-binding site,
boundtoanenzyme,theactivesitesofcatalyticallyactivegly- GRRDKQH, at positions 143–149. Interestingly, this domain
cosidehydrolasesdistortthepyranoseringinthe(cid:3)1binding
followsaconsensusproteinsequence,XBBXBX(whereBisa
subsiteintoalessenergeticallyfavorableconformation,suchas
basicresidueandXisanynon-basicaminoacid),thatisnoted
aboatorskewconformation(62–65),primingthesubstratefor
for its ability to recognize polyanions like heparin (67). In all
hydrolytic cleavage. Interestingly, the chitohexaose ligand
fourcases,heparinrecognizedthebindingsitewithin25nsof
bound in the primary binding site of YKL-40 exhibits a boat
MDsimulation(Fig.5),occasionallyvisitingothermoderately
conformation despite not being catalytically active (10). This
basicsurfacelocationsbeforelocalizingaroundtheGRRDKQH
suggeststhatthesugardistortioninthe(cid:3)1bindingsitecon-
motif. The strong electrostatic interaction arose from the
tributestoligandbindingaswellascatalysis,becausethereisno
dynamicformationofsaltbridgesbetweeneitherthesulfateor
evolutionary requirement to overcome an activation energy
thecarboxylgroupsoftheheparinoligosaccharideandtheside
barrierincatalyticallyinactivelectins.Arecentstudyofaho-
mologous chitinase suggested that (cid:3)1 pyranose relaxation chainsofthebasicaminoacids(supplementalTableS3).Lys155
andLys193alsoexhibitlarge,favorableinteractionswithhepa-
reducesbindingaffinityandaffordstheligandmoreflexibility
rin, interacting with the opposite end of the polysaccharide
andentropicfreedom(66),whichisconsistentwithourfind-
ligand as it binds at the surface. Coupled with experimental
ingsfromthe250-nsMDhere.
PutativeHeparin-bindingSite—Despitethefactthatthehep- observationofheparinaffinity,ourMDsimulationssuggesta
arinoligomercouldnotbeaccommodatedbytheYKL-40bind- non-specific, surface-mediated binding interaction between
ingcleft,MDsimulationsdosuggestthattheoligomerinteracts YKL-40andtheextensivelysulfatedheparinoligomer(10,11).
with the surface of YKL-40 at a putative heparin-binding site Althoughtheunsulfatedvariant,heparansulfate,didnotvisit
(Fig. 1). After ejection from the primary binding site, the oli- the heparin-binding site, chondroitin sulfate also attached to
gomerspontaneouslybindstotheYKL-40heparin-bindingsite theputativeheparin-bindingsiteinasimilarfashiontoheparin.
(supplemental Movie S1). To address the significance of this Giventhechemicalsimilarityoftheseglycosaminoglycans,i.e.
unanticipated event, we performed three additional indepen- highly sulfated and negatively charged, we anticipate the
FEBRUARY17,2017•VOLUME292•NUMBER7 JOURNALOFBIOLOGICALCHEMISTRY 2629
IdentifyingthePhysiologicalLigandofYKL-40
TABLE1
EnergeticcomponentsofthefreeenergyofligandbindingtoYKL-40
System (cid:2)G (cid:2)G (cid:2)G (cid:2)G (cid:2)G (cid:2)G
repu disp elec rstr Tot b
kJmol(cid:3)1 kJmol(cid:3)1 kJmol(cid:3)1 kJmol(cid:3)1 kJmol(cid:3)1 kJmol(cid:3)1
YKL-40(cid:2)Cellohexaose 379.5(cid:5)3.8 (cid:3)382.3(cid:5)2.1 (cid:3)306.6(cid:5)2.2 (cid:3)1.1 (cid:3)310.5(cid:5)3.6 (cid:3)12.6(cid:5)3.7
Cellohexaose 316.2(cid:5)1.8 (cid:3)287.4(cid:5)1.3 (cid:3)326.7(cid:5)1.6 0 (cid:3)297.9(cid:5)2.8
YKL-40(cid:2)Chitohexaose 535.9(cid:5)11.7 (cid:3)538.9(cid:5)4.2 (cid:3)407.7(cid:5)7.5 (cid:3)5.5 (cid:3)416.2(cid:5)11.2 (cid:3)63.3(cid:5)12.6
Chitohexaosea 329.6(cid:5)4.5 (cid:3)306.2(cid:5)3.0 (cid:3)376.3(cid:5)3.4 0 (cid:3)352.9(cid:5)5.9
YKL-40(cid:2)Hyaluronan 438.1(cid:5)2.8 (cid:3)439.5(cid:5)1.5 (cid:3)1395.9(cid:5)4.2 (cid:3)1.3 (cid:3)1398.6(cid:5)4.3 (cid:3)106.8(cid:5)4.6
Hyaluronan 333.1(cid:5)1.4 (cid:3)306.0(cid:5)1.3 (cid:3)1318.9(cid:5)1.5 0 (cid:3)1291.8(cid:5)2.5
aHamreetal.(68).
XBBXBXmotifmayalsoroutinelyappearinchondroitin-bind- charideligandcapableofbindingintheYKL-40bindingcleft
ingproteins. willlikelyexhibitasimilarlyfavorableWCAbindingfreeenergy
Polysaccharide Ligand Binding Affinity—Each of the three component. In the following section, we expand upon the
polysaccharidesmaintainingcontactwiththeprimarybinding molecularlevelinteractionsthatcontributetopolysaccharide
siteofYKL-40,cellohexaose(orlikelyanyglucosederivative), bindingaffinityinYKL-40.
chitohexaose, and hyaluronan, are feasible ligands. However, Based on these results, it is unlikely that a cello-oligomer
freeenergycalculationssuggestthathyaluronanmaypreferen- wouldbindinthecleftofYKL-40overachito-oligomer,and
tially bind with YKL-40 if chitin is not present in the human thus, although there is potential for YKL-40 to bind a cello-
bodyasaresultoffungalinfection.Theabsolutefreeenergiesof oligomerorglucose,itwouldnotbeinhibitory.Hyaluronan,on
bindingcellohexaose,chitohexaose,andhyaluronantoYKL-40 theotherhand,likelycompeteswithchito-oligomersinbind-
were (cid:3)12.6 (cid:5) 3.7, (cid:3)63.3 (cid:5) 12.6, and (cid:3)106.8 (cid:5) 4.6 kJ/mol, ing,whichisdueinlargeparttotheelectrostaticfavorabilityof
respectively. Repulsive, dispersive, and electrostatic compo- thechargedsidechainsofhyaluronanintheYKL-40binding
nentsofthefreeenergychangesaretabulatedinTable1.Con- cleft.Clinicaldatasupporthyaluronanasabiomarkerforcan-
vergence assessment of the free energy calculations has been cer prognosis and inflammation (32, 70), the same events in
provided in supplemental Fig S3. We recently calculated the which YKL-40 appears at elevated serum levels (5). To our
freeenergyofsolvationforchitohexaoseaspartofastudyon knowledge,therearenostudiesevaluatingtheco-existenceof
family18chitinases(68);thisvaluehasbeenusedinourcalcu- YKL-40 and hyaluronan. The cell receptor protein CD44 has
lationofchitohexaosebindingaffinitytoYKL-40forcomputa- been implicated in hyaluronan binding interactions and is
tionalefficiency.Themethodsusedtocalculatesolvationfree also involved in confounding scenarios, both aggravating and
energyofchitohexaosewereidenticaltothosedescribedhere. improvinginflammation(71).SequencealignmentofYKL-40
Furthermore, the binding free energy of chitohexaose to with the hyaluronan-binding domain of human CD44 (72),
YKL-40isingoodagreementwiththatofhomologousfamily18 usingBLASTP2.3.0(73),showsnohomology,furthersuggest-
chitinases,despitemutationofthecatalyticmotifinthelectin. ingthatthisYKL-40-hyaluronanbindingisdifferentfrompre-
Chitohexaoseandcellohexaosearebothneutralligandsbut viouslyknownhyaluronan-bindingproteins(74).
display a significant difference in binding affinity to YKL-40. PolysaccharideBindingDynamics—YKL-40ishighlyhomo-
Electrostaticinteractionsappeartobeoneofthemoresignifi- logous with carbohydrate-active enzymes found in glycoside
cantcontributorstotheenhancedaffinityofchitohexaoseover hydrolasefamily18(12,75).Despitelackingcatalyticability,the
cellohexaose(Table1).Forcellohexaose,thechangeintheelec- primary polysaccharide binding site of YKL-40 exhibits
trostatic component of binding free energy was unfavorable remarkablesimilaritytothesefamily18chitinases.Assuch,one
(20.1 (cid:5) 2.7 kJ/mol), whereas the same component for chito- may reasonably expect that ligand binding within this family
hexaose was energetically favorable ((cid:3)31.4 (cid:5) 8.2 kJ/mol). In willdemonstratesimilartrends,regardlessofevolutionaryori-
thecaseofhyaluronan,electrostaticinteractionsplayaneven gin. Indeed, we observe that chitohexaose, cellohexaose, and
greater role in enhancing affinity of the ligand for YKL-40 hyaluronan binding in the primary binding site of YKL-40
((cid:3)77.0 (cid:5) 4.5 kJ/mol). This increasing electrostatic contribu- follow a general pattern common to carbohydrate-active
tionisreflectiveofincreasingnumberofelectronegativeatoms enzymes. Namely, that ligand binding interactions are medi-
inthesidechainsofcarbohydratesaswegofromcellopentaose ated by carbohydrate-(cid:6)stacking interactions with aromatic
tochitohexaosetohyaluronan.Weobservenosignificantdif- residues, and hydrogen bonding interactions are critical to
ferencesincellohexaose,chitohexaose,orhyaluronanbinding overallligandaffinityandstability.Weinvestigatethesetrends
to YKL-40 arising from Weeks-Chandler-Anderson (WCA) quantitativelythroughanalysisoftheMDsimulationtrajecto-
dispersionandrepulsion(Table1).Thisislargelyafunctionof ries,includingrootmeansquaredeviation(RMSD)ofthepro-
themolecularsimilarityofthepyranoseringscomprisingthe tein,rootmeansquarefluctuation(RMSF)ofboththeprotein
monomericunitsofthreeoligosaccharides(Fig.2).Thepyra- and the ligands over the course of the simulation, hydrogen
noseringsofcarbohydratesboundintheactivesitesofglyco- bondinganalysis,degreeofsolvationoftheligand,andinterac-
sidehydrolases,andbyextensionthebindingcleftsoflectins, tionenergyoftheligandwiththeprotein.
form carbohydrate-(cid:6)stacking interactions with surrounding Cellohexaose,chitohexaose,andhyaluronanbindinginthe
aromaticresiduesalongtheclefts(69).InYKL-40,thesestack- primary YKL-40 binding site did not adversely affect protein
inginteractionsareformedinthe(cid:3)3and(cid:3)1bindingsiteswith dynamics.Ineachcase,bindingthepolysaccharideliganddid
residuesTrp31andTrp352,respectively.Naturally,anypolysac- notsignificantlydisturbtheproteinbackbone(i.e.proteinfold),
2630 JOURNALOFBIOLOGICALCHEMISTRY VOLUME292•NUMBER7•FEBRUARY17,2017
IdentifyingthePhysiologicalLigandofYKL-40
luronanisasstable,ifnotmoreso,aschitohexaoseinthepri-
marybindingsite(Fig.6a);however,cellohexaoseappearstobe
morestablerelativetothetwootherligandsattheendsofthe
bindingcleft.Thislatterfindingisafunctionofthelengthofthe
sidechainsattachedtothepyranoseringsofeachoftheligands.
Ofthethreecarbohydrates,thecello-oligomerhastheshortest
sidechains,andthus,theligandfluctuateslessbecauseitdoes
not need to rearrange as significantly to induce binding. As
shownabove,thisdoesnotnecessarilycorrespondtothemost
thermodynamicallypreferentialligand,andlowerRMSFcould
also be interpreted hypothetically as loss of translational and
conformational freedom, resulting in unfavorable entropic
contribution.
The hydrogen-bonding partners of chitohexaose, cello-
hexaose,andhyaluronanarequitedifferent,largelyasaresult
of the conformational change of hyaluronan (supplemental
TableS4).Definingahydrogenbondasapolaratomwithin3.4
Å and 60° of a donor, we identified the formation of donor-
acceptorpairsandpercentoccupancyofthesehydrogenbonds
between the protein and each carbohydrate moiety over the
courseofthe250-nsMDsimulations(supplementalTableS4).
Asdescribedabove,hyaluronanformedasharpVshapeinthe
polysaccharidebindingcleft,whichminimizedsterichindrance
and, in turn, modified accessible hydrogen bonding partners
relativetochito-andcellohexaose.Hydrogenbondsatthe(cid:2)1
site, between glucuronic acid and Asp207, Arg263, and Tyr141,
stabilized the hyaluronan conformation (supplemental Table
S4).Inthe(cid:3)1subsite,chitohexaoseprimarilyhydrogenbonds
withthesidechainofTyr206andthemainchainofTrp99.Inthe
casesofbothcellohexaoseandhyaluronan,theinteractionwith
Tyr206wasabolishedandinsteadsupplementedbyTrp99alone.
Inthe(cid:3)2subsite,theoxygenofthechitohexaoseacetylformsa
long-livedhydrogenbondwiththeindolenitrogenofthebur-
iedTrp352;neitherhyaluronannorcellohexaoseinteractwith
the (cid:3)2 site through this tryptophan. Rather, Trp31, which
stackswiththepyranoseinthe(cid:3)3subsite,actsasahydrogen
donortothe(cid:3)2subsiteglucuronicacidsidechainofhyaluro-
nan.Inthecaseofcellohexaose,themainchainofasolvent-
FIGURE6.a,RMSFofthepolysaccharideligandsonaper-binding-subsite exposed asparagine, Asn100, almost exclusively mediates
basis.Theerrorbarswerecalculatedusingblockaveragingover2.5ns.b, hydrogenbondinginthe(cid:3)2site.The(cid:2)2,(cid:3)3,and(cid:3)4binding
averagenumberofwatermoleculeswithin3.5Åofeachligandmonomer.
subsites exhibit less frequent hydrogen bonding between the
Theerrorbarsrepresentonestandarddeviation.
ligandandtheprotein,andthereislittleconsistencyinbonding
andtheligandremainedrelativelyunperturbedoverthecourse partnersacrossligands.Certainly,thesevariationswillmanifest
ofthesimulation.TherelativelyconsistentRMSDofthepro- in enthalpic contributions to ligand binding, because even a
tein backbones suggests that the simulations reached a local singlehydrogenbondmayaccountfor4–29kJ/molofbinding
equilibrium(supplementalFig.S4a).AswiththeRMSDcalcu- freeenergyinbiologicalsystems(76,77);suchislikelythecase
lation, the RMSF of the protein backbone suggests that the for cellohexaose and chitohexaose binding to YKL-40, where
bindingofpolysaccharidesdoeslittletodisturbtheoverallpro- the latter exhibits both greater hydrogen bonding capability
tein conformation (supplemental Fig. S4b). Additionally, we andamorefavorablebindingfreeenergy.
didnotobservesignificantconformationalchangesinthecar- Keyaromaticresidues,Trp31,Trp99,andTrp352,playasig-
bohydrate-bindingsiteofYKL-40beforeorafterligandbinding nificant role in binding all three oligosaccharides. Notably,
(supplementalFig.S5).Moredetailsofproteindynamicsand these tryptophans are conserved in other lectins, including
conformationalchangeshavebeenprovidedinthesupplemen- mammaryglandprotein(MGP-40)andmammalianlectinYm1
taltext. (20, 78). According to previous structural studies, these aro-
TheRMSFoftheligand,averagedover250nsasafunctionof matic residues form hydrophobic stacking interactions with
binding site, provides a measure of relative ligand stability. pyranose moieties at the (cid:3)3, (cid:2)1, and (cid:3)1 binding subsites,
Error was estimated by block averaging over 2.5-ns blocks. respectively (11). As mentioned above, this carbohydrate-(cid:6)
Ligandstabilityoverthecourseofthesimulationsuggestshya- stackingwasobservedacrossthethreepolysaccharideligands
FEBRUARY17,2017•VOLUME292•NUMBER7 JOURNALOFBIOLOGICALCHEMISTRY 2631
IdentifyingthePhysiologicalLigandofYKL-40
FIGURE7.Nativecontactanalysisofeachcollagen-likepeptidemodelbindingtoYKL-40atsiteA.Thecolorscalerepresentsthenormalizedfrequency
(i.e.fractionalpercentageofframesinwhichthecontactwasformed)oftherespectiveYKL-40residueasanativecontact.Anativecontactwasdefinedasany
timeacollagenresiduewaswithin12ÅofaYKL-40residue,wheredistancewasdefinedbycenterofgeometryofagivenresidue.Onlyframesfromthelast100
nsofsimulation,followingaperiodofequilibration,wereconsideredinthisanalysis.
asaresultofthechemicallysimilarcarbohydrate“backbone”of passesasmanypotentialbindingmodesasfeasibletodescribe
pyranose rings. However, at the (cid:2)1 site of hyaluronan, the protein-protein binding dynamics and relative affinity of
stacking interaction with Trp99 was not maintained. Instead, YKL-40forcollagen.
prominenthydrogenbondingforcesthe(cid:2)1pyranoseringinan LigandBindingDynamicsandComparisonofModelCol-
orientation that is perpendicular to aromatic Trp99 (supple- lagen-like Peptides—Dynamics of the collagen-like peptide
mentalFig.S6).Nevertheless,thesimilarityinWCAcontribu- ligandsvarieswithbothbindingsiteandthepitchofthetriple
tionstobindingfreeenergyforallthreepolysaccharidessug- helix.Rootmeansquaredeviationillustratestherelativestabil-
geststhatthis(cid:2)1sitestackinginteractionweaklycontributes ityofeachcollagenpeptideineachofthetwobindingsites,A
totheoverallbindingfreeenergy. andB(supplementalFig.S7).Althoughthemoleculardocking
ThedegreetowhichthebindingcleftofYKL-40wasacces- resultsinveryclosecontactbetweencollagenandYKL-40(Fig.
sibletowatermoleculesdidnotchangesignificantlywiththe 4), such that collagen appears to be almost buried in the pri-
bound polysaccharide. The degree of ligand solvation was marycarbohydrate-bindingsiteofYKL-40,minimizationand
determinedbycalculatingtheaveragenumberofwatermole- MDsimulationresultsintheslightriseandshiftintheposition
culeswithin3.5Åofagivenpyranoseringoverthecourseofthe ofcollagenforeverymodelatbindingsiteA.Eachofthefour
simulations(Fig.6b);errorisgivenasonestandarddeviation. ligandsmaintainsassociationwiththebindingsiteAoverthe
Chitohexaoseandcellohexaosedisplaysimilardegreesofsol- courseof250ns,althoughwithslightlydifferentprotein-pro-
vationacrossthelengthofthecleft.Hyaluronanallowsamod- tein contacts with YKL-40 (Fig. 7). Native 1CAG, 1BKV, and
erateincreaseindegreeofsolvationofthecleftbycomparison 1Q7Dattainedrelativestabilityinapositionnotsignificantly
tochitohexaose,acrossthe(cid:3)3and(cid:2)1subsites,whereitssharp differentfromtheinitialdockedposition,butthe1CAGpep-
Vshapeagaincontributestovariationinbehavior.Giventhe tide (supplemental Movie S3), with disrupted helical content
similarity in solvent accessibility within the binding cleft resultingfromtheglycinetoalaninemutation,requiredanadjust-
regardless of ligand, it is unlikely that entropic contributions mentinpitchbeforeassociatingwithYKL-40.Thisrelativechange
fromsolvationplayaroleintheobserveddifferencesinligand in position is shown in the RMSD of the peptides during first
bindingfreeenergy. 50–100nsbeforestabilization(supplementalFig.S7a).Binding
Protein-Protein Binding in YKL-40—Based on biochemical site B accommodates helical pitches of 7/2 collagen peptides,
characterization,itisclearthatYKL-40functionallyinteracts becausenative1CAGand1Q7DassociatedwithYKL-40with
withcollagen.Forexample,Biggetal.(9)uncoveredtheability very little change in orientation relative to the initial docked
ofYKL-40tospecificallybindtypesI,II,andIIIcollagenfibers, positions.The1CAGligandwasexpelledfrombindingsiteB,as
andIwataetal.(79)recentlydiscoveredthatYKL-40secreted wasthesomewhatimperfect10/3-pitched1BKVpeptide.This
by adipose tissue inhibits degradation of type I collagen by suggeststhatYKL-40mayavoidphysiologicalinteractionswith
matrix metalloproteinase-1 and further stimulates the rate of certaincollagenfibrildomains,especiallythosehavingimper-
type I collagen formation. However, a lack of structural evi- fecthelicalpitches.Theintegrin-bindingcollagen-likepeptide
dencehasprecludeddevelopmentofanunderstandingofthe 1Q7Ddemonstratedthegreateststabilityamongcollagenpep-
molecularnatureoftheseinteractions.Usingmoleculardock- tidesinbothbindingsites(supplementalFig.S7)andformed
ing,MDsimulation,andfreeenergycalculations,wedescribe morenativecontactswithYKL-40thantheotherthreecollagen
interactions of four collagen-like peptides with two putative peptides at binding site A (Fig. 7). We anticipate that the
protein-binding sites along the surface of YKL-40. The selec- GFOGERmotifplayssubstantialroleinmediatingtheinterac-
tionofmodelpeptides,aswellasmultiplebindingsites,encom- tionofthiscollagenpeptidewithYKL-40.
2632 JOURNALOFBIOLOGICALCHEMISTRY VOLUME292•NUMBER7•FEBRUARY17,2017
IdentifyingthePhysiologicalLigandofYKL-40
Examiningthenumberofnativecontactsbetweeneachcol-
lagenpeptideandbindingsiteAofYKL-40revealsseveralcom-
moninteractionsitesmediatecollagenbindingandhelpsnar-
row down key regions of interest (Fig. 7). YKL-40 residues
69–71, 98–108, 205–215, and 230–235 interact with all four
collagenpeptidesandlikelycontributetobindingaffinity,aswe
willdiscussbelow.TheregionofYKL-40betweenresidues179
and189associateswithnative1CAG,1BKV,and1Q7D,butnot
withtheoriginal1CAG,asthispeptidewithrelaxedsymmetry
neededtoadjustitspositionfromdockedconformationtosta-
bilize the interactions. The 1Q7D model formed the greatest
number of interactions with YKL-40 residues relative to the
otherthreemodels.Similarnativecontactanalysisforbinding
siteBshowsthatevenN-terminalandC-terminalresiduesof
YKL-40areinvolvedincollagenbindingatbindingsiteB(sup-
plementalFig.S8).Itshowsthat,unlikebindingsiteA,thereis
littledifferenceinthenumberofinteractionsofmodel1Q7D
and native 1CAG collagen peptide with the binding site B of
YKL-40.
To better understand the interactions collagen makes with
YKL-40, identified through the native contact analysis, we
calculatedelectrostaticandvanderWaalsinteractionener-
giesofeachYKL-40residuewitheachcollagenpeptideover
the250-nsMDsimulations(supplementalTableS5).Visual
inspectionofthesimulationsrevealsaromaticresiduesinthe
binding sites, such as Trp212 and Trp99 in binding site A and
Phe49 in binding site B, were involved in aromatic-proline
stacking interactions with the collagen triple helices. Such
interactions are favorable, occurring because of both hydro-
phobiceffectsandinteractionbetweenthe(cid:6)aromaticfaceand
thepolarizedC-Hbonds(80).Thisisillustratedinthevander
Waalscomponentoftheinteractionenergy,whereatbinding
siteA,Trp69,Trp71,Trp99,Trp212,andPhe234showsubstantial
favorableinteractionwithcollagenpeptides,althoughthecon-
tribution varies with each collagen peptide (supplemental
TableS5).Additionally,acidicandbasicresiduesoftheinteg-
rin-bindingGFOGERmotiffromcollagen-likepeptide1Q7D FIGURE8.CollagenbindingwithYKL-40.a,saltbridgesformedbetween
formionicinteractionswiththecounter-ionicaminoacidsof the1Q7Dcollagenpeptide(greencartoon)andbindingsiteA(graysurface).b,
saltbridgeinteractionsofthe1Q7Dcollagenpeptide(cyancartoon)with
YKL-40, also known as salt bridges. Specifically, 1Q7D forms
bindingsiteB(graysurface).c,bindingfreeenergyobtainedfromumbrella
threesaltbridgesatbindingsiteAandonesaltbridgeatbinding sampling MD simulations of the YKL-40-collagen peptide systems, inter-
siteB(Fig.8).AtsiteA,Arg105,Asp207,andArg263ofYKL-40 pretedasnegativeofPMFtodecouplethepartners.Thecollagenpeptidesin
questionare1Q7D(atbothsites)and1CAG(atsiteAonly).Thefreeenergyis
interact with Glu(a11), Arg(c12), and Glu(c11) of 1Q7D,
shownasafunctionofnormalizedreactioncoordinate,wherethereaction
respectively,wherethea,b,orcintheparenthesescorresponds coordinateisfractionofnativecontacts.
to one of the three strands of the collagen model. Notably,
Glu(a11),Arg(c12),andGlu(c11)belongtotheGFOGERinteg- staticinteractionenergies;supplementalTableS5),althoughas
rin-bindingmotif.AtsiteB,Lys23ofYKL-40formsasaltbridge aresultofhydrogenbondingratherthansaltbridgeformation.
with Glu(a11) of 1Q7D. As anticipated, the GFOGER motif WenotethattheinteractionenergiesofYKL-40residueswith
playedasubstantialroleintheinteractionofthiscollagenpep- residuesofeachcollagenmodelpeptidearenotconservedasa
tide with YKL-40, but its role was different from that of the resultofdifferencesinthecollagensequences,particularlyin
integrinbindingmechanism,whichfurtherinvolvescoordina- themiddleregionsconsistingofdifferentimino-triplets.
tionofametalion(42).Nevertheless,saltbridgesandhydro- From MD simulation, we observe substantial hydrogen
phobiccontactsareveryimportantinbothcases,significantly bondingbetweenthecollagenpeptidesandYKL-40acrossthe
contributing to the electrostatic component of the binding lengthofeachbindingsite,whichcontributestooverallstabil-
affinity of this collagen peptide relative to collagen peptides ityandbindingaffinity.Thehydrogenbondinganalysisforthe
lackingacidicorbasicaminoacids(e.g.native1CAG)(supple- collagen-YKL-40systemswasperformedasdescribedabovefor
mental Table S5). The hydroxyl oxygens of hydroxyprolines the polysaccharide ligands; pairs exhibiting greater than 10%
from 1CAG and native 1CAG appear to be involved in ionic occupancy over the simulation are reported individually in
interactions with acidic YKL-40 residues (favorable electro- supplemental Table S6. The YKL-40 residues responsible for
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