Hauv Silico Kev Ntsuam Xyuas Ntawm Drosophila Parasitoid Venom Peptide Qhia Txog Feem Ntau Ntawm Cation-Polar-Cation Clip Motif hauv Knottin Proteins Part 1

Abstract:

Raws li generalist parasitoid wasps, Leptopilina heteroatom muaj kev vam meej ntawm ntau hom txiv hmab txiv ntoo yoov ntawm genus Drosophila. Cov kab mob parasitoids tsim tshwj xeeb ntau lub tswv yim extracellular vesicle (EV) zoo li cov qauv hauv lawv cov venom. Kev soj ntsuam Proteomic tau txheeb xyuas ntau yam kev tiv thaiv kab mob sib txuas nrog cov protein, suav nrog knottin peptide, LhKNOT, uas muaj cov txheej txheem khaws cia inhibitor cysteine ​​knot (ICK) fold, uas muaj nyob hauv cov proteins los ntawm ntau cov taxa. Peb cov txheej txheem thiab kev txheeb xyuas ntawm LhKNOT's 36-residue core knottin fold tau qhia tias ntxiv rau knottin motif nws tus kheej, nws kuj muaj Cation-Polar-Cation (CPC) clip.

Inhibitor cysteine ​​​​yog ib yam khoom ntuj siv los tiv thaiv oxidative puas tsuaj thiab txhawb kev loj hlob ntawm tes. Rau kev tiv thaiv kab mob, lub luag haujlwm ntawm cysteine ​​yog qhov tseem ceeb hauv kev txhim kho lub cev tiv thaiv kab mob thiab tiv thaiv kev mob thiab kis kab mob.

Nyob rau hauv xyoo tas los no, ntau thiab ntau cov kev tshawb fawb tau pom tias cysteine ​​tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob. Nws tuaj yeem txhawb kev loj hlob ntawm cov hlwb tiv thaiv kab mob, txhim kho lub cev tiv thaiv kab mob, thiab txo qhov kev pheej hmoo ntawm kev kis kab mob. Tsis tas li ntawd, cysteine ​​tuaj yeem txo qis kev tsim cov dawb radicals thiab txhim kho kev noj qab haus huv tag nrho ntawm lub cev tiv thaiv kab mob, yog li txhim kho lub cev tiv thaiv kab mob.

Txawm hais tias cov nyhuv ntawm cysteine ​​​​ntawm kev tiv thaiv kab mob tseem ceeb heev, thaum siv cov tshuaj inhibitor cysteine, nws yuav tsum tau siv nrog ceev faj. Vim tias cov tshuaj inhibitor cysteine ​​feem ntau yog siv los kho qee yam kab mob, xws li kab mob plawv, mob caj dab, thiab lwm yam, yog tias raug tsim txom, nws yuav cuam tshuam rau kev tiv thaiv kab mob thiab ua rau lub cev tsis muaj zog, yog li ua rau muaj kev pheej hmoo kis mob.

Hauv kev xaus, muaj kev sib raug zoo ntawm cov inhibitor cysteine ​​thiab kev tiv thaiv kab mob. Txawm hais tias inhibitors ntawm cysteine ​​tuaj yeem txhawb kev noj qab haus huv ntawm lub cev, lawv kuj yuav tsum tau siv raws li kev qhia ntawm tus kws kho mob, thiab kev saib xyuas yuav tsum tau them nyiaj rau kev tswj hwm tus nqi tsim nyog los tswj kev noj qab haus huv ntawm lub cev. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev tiv thaiv. Cistanche tuaj yeem txhim kho kev tiv thaiv zoo, vim tias cov hmoov tshauv muaj ntau yam khoom xyaw lom neeg, xws li polysaccharides, ob lub nceb, Huang Li, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb nqa ntau yam kev tiv thaiv kab mob. cell zoo li cov hlwb, ua kom lawv lub cev tiv thaiv kab mob.

Nyem qhov txiaj ntsig kev noj qab haus huv ntawm cistanche

CPC clip motif yog xav kom pab txhawb kev ua haujlwm ntawm cov tshuaj tua kab mob hauv heparin-binding proteins. Kuj ceeb tias, feem ntau ntawm ICKs tau sim kuj muaj CPC clip motif, suav nrog 75 bona fide cog thiab arthropod knottin proteins uas sib koom ua ntu zus thiab / lossis cov qauv zoo sib xws nrog LhKNOT. Zoo li LhKNOT thiab lwm yam 75 knottin proteins, txawm tias Drosophila Drosomycin antifungal peptide, ib hom kab mob canonical ntawm yoov's TollNF-kappa B tiv thaiv kab mob, muaj qhov CPC clip motif. Ua ke, peb cov txiaj ntsig tau qhia tias muaj peev xwm tiv thaiv tau rau tus kab mob LhKNOT.

Qhov tshwm sim ntawm CPC clip motif, intrinsic rau cysteine ​​knot nyob rau hauv lub knottin proteins tshuaj ntsuam xyuas ntawm no, qhia hais tias qhov tshwm sim 3D topology yog ib qho tseem ceeb rau lawv biochemical functions. CPC clip yuav yog ib qho kev txuag zoo heev uas pom muaj nyob rau hauv ntau cov proteins sib txawv nrog cov ntaub ntawv qhia txog heparin binding muaj peev xwm, suav nrog amyloid proteins. Knottins yog lub hom phiaj rau kev txhim kho tshuaj kho mob, thiab kev nkag siab rau lawv cov qauv-kev sib raug zoo yuav ua rau kev tsim tshuaj tshiab.

Ntsiab lus:

Host-parasite; Drosophila; Leptopilina; cysteine ​​pob txha; knottin quav; Cation-Polar-Cation clip; antimicrobial peptide; heparin-binding motif; mini proteins.

1. Taw qhia

Hauv kev sib cuam tshuam ntawm tus tswv-parasite, tus kab mob yuav tsum muaj kev tawm tsam thaum tib lub sijhawm ua kom tus tswv muaj sia nyob kom txhawb nqa cov xeeb ntxwv [1,2]. Xws li cov hom phiaj tsis sib haum xeeb tau ua rau muaj kev hloov pauv ntawm cov xwm txheej virulence nrog ntau cov tswv yim molecular thaum tswj hwm tus tswv tsev tshwj xeeb. Parasitic Hymenoptera ua rau ntau tshaj li ib lab hom, muaj thoob plaws ntiaj teb, thiab parasitize ntau yam arthropods, feem ntau yog kab [3].

Wasps tawm tsam kab hosts tau tsim ntau yam kev coj tus cwj pwm thiab biochemical cov tswv yim rau kev ua tiav thiab nyob twj ywm nyob rau hauv qhov kev sib koom ua ke nrog lawv cov tswv [4–7].

Leptopilina / Drosophila system yog ib qho qauv tshiab rau kev nkag siab lub hauv paus molecular ntawm cov lus teb los tiv thaiv kab mob thiab cov tswv yim kab mob phem [8]. Txhawm rau tiv thaiv kev tiv thaiv tus tswv tsev, Leptopilina cov poj niam qhia venom yam rau hauv lawv cov larval hosts thaum oviposition [4,9,10]. Cov txheej txheem tiv thaiv kab mob hauv lub cev zoo hauv Drosophila [11–13] muab cov ntsiab lus tseem ceeb los kawm txog qhov cuam tshuam ntawm cov kab mob venom no.

Kev kis kab mob los ntawm tus kws kho mob tshwj xeeb L. boulardi activates lub caj npab humoral thiab cellular tiv thaiv kab mob uas tswj los ntawm yoov txoj kev txuag tus xov tooj hu-NF-κB. Kev qhia ntawm cov tshuaj tiv thaiv kab mob peptide (AMP) cov noob xws li drosomycin yog qhib tom qab L. boulardi kab mob [14]. Kev xa xov tooj-NF-κB kuj tseem tswj cov ntshav cell faib thiab kev loj hlob [15,16], cov kauj ruam uas tswj encapsulation thiab tuag ntawm qe qe [17,18].

Hauv qhov sib piv, kev kis kab mob los ntawm tus kws tshaj lij L. heteroatom suppresses ob txoj hauv kev tiv thaiv kab mob [9,14], thiab yuav luag tag nrho cov qe ntshav ntawm cov qe ntshav raug rhuav tshem los ntawm cov khoom hauv cov kab mob venom [19,20].

Hauv txoj kev tshawb no, peb txheeb xyuas cov qauv theoretical ntawm ib qho L. heteroatom peptide, hu ua LhKNOT, txheeb xyuas nyob rau hauv organelle-zoo li secretions hauv L. heteroatom venom tab sis tsis nyob rau hauv L. boulardi venom [21]. Cov kab mob tiv thaiv kab mob no suav nrog ntau dua 350 cov proteins thiab muaj cov kab mob proteomic ntawm eukaryotic microvesicles thiab ntau ntau ntawm kev tiv thaiv kab mob ntsig txog cov protein [21-23].

LhKNOT txais yuav "Inhibitor Cysteine ​​Knot" (knottin) motif [24]. Lub knottin motif yog dav kev txuag nyob rau hauv ntau yam ntawm taxa, los ntawm cov nroj tsuag [25,26] mus rau qwj [27]. Cov peptides me me no (<60 residues) are functionally diverse. Knottins are used offensively, in spider toxins [28] and cone snail venom [27], as well as defensively, e.g., in wound healing activities of cacti [26], and antimicrobial host defense in plants [25,26,29] and invertebrates [30].

Qhov ntau yam ntawm knottin kev ua si yog ntaus nqi rau tag nrho cov qauv ntawm nws cov quav, uas tsuas yog nyob ntawm ib qho kev txuag motif ntawm rau cysteine ​​residues uas yuav siv sij hawm kom zoo dua ntawm cov cysteine ​​'lub peev xwm tshwj xeeb los tsim cov choj disulfide. Lub knottin motif tshwm sim thaum pom cov cysteine ​​seem nyob rau hauv C1-C2-C3-C4-C5-C6 qauv (qhov "-" sawv cev rau ntau yam ntau variable residues).

Cov txuas hniav disulfide tau tsim nyob rau hauv ib qho kev txuag zoo sib xws (C1-C4, C2-C5, thiab C3-C6), ua rau C1-C4 thiab C{{ 8}}C5 tsim lub macrocycle, thaum C3-C6 tshwm sim hauv macrocycle no, ua rau "knotted" topology. Raws li qhov knotted topology no feem ntau hinges rau kev txuag ntawm rau cysteine ​​residues, lub knottin motif tso cai rau kev hloov pauv tseem ceeb hauv tag nrho cov kab ke [30]. Kev sib xyaw ua ke ntawm lawv qhov me me, kev ua siab ntev ntawm qhov sib txawv ntawm qhov sib txawv, thiab pom cov qauv kev ruaj ntseg tau ua rau knottins nrov scaffold rau kev tsim tshuaj [30], muaj peev xwm txawm rau kev kho mob qog noj ntshav [31].

Ntau cov kab ntawm kev sim ua pov thawj pom lossis qhia txog kev muaj peev xwm tiv thaiv kab mob rau cov kab mob sib txawv [25,26,29,30,32,33] lossis gated ion channel kev sib cuam tshuam [28,34], txawm hais tias cov txheej txheem molecular uas cov haujlwm no ua tsis tau. nkag siab zoo. Tsis tas li ntawd, nws tsis paub yog tias cov tshuaj tiv thaiv kab mob no sib koom ua ke ntawm kev ua haujlwm.

Muab nws qhov kev tshawb pom los ntawm kev tiv thaiv kab mob los ntawm cov kab mob yoov, peb lub hom phiaj yog (a) txheeb xyuas cov kab mob LhKNOT los ntawm ntau hom kab mob, suav nrog L. heteroatom, L. boulardi, thiab Ganaspis parasitoids, uas tawm tsam Drosophila thiab (b) txheeb xyuas ib qho. tshiab cov qauv motif (s) tam sim no nyob rau hauv lub paub los yog tshiab LhKNOT homologs nrog kev cia siab tias qhov kev kwv yees ntawm xws li ib tug conserved motif yuav qhia tau hais tias ua tau hauj lwm rau LhKNOT thiab qhia yav tom ntej kev tshawb fawb.

Peb qhia tau hais tias tus qauv theoretical ntawm LhKNOT muaj cov cim tseem ceeb ntawm knottin quav nrog peb cov nplooj ntawv antiparallel beta, txwv los ntawm cov choj disulfide. Tus qauv LhKNOT muaj CPC clip, muaj peev xwm cuam tshuam nrog heparin, tus nqi tsis zoo glycosaminoglycan (GAG) ntawm tus nqi kho mob tseem ceeb. LhKNOT kuj muaj peev xwm cuam tshuam nrog lwm cov GAGs, xws li keratan sulfate (GAG uas yog sulfated heev, zoo ib yam li heparin) thiab hyaluronic acid (ib tus neeg sawv cev GAG uas tsis tas yuav muaj sulfation) [35].

Tsis tas li ntawd, cov qauv homologs ntawm LhKNOT (piv txwv li, bona fide knottin motif proteins) kuj muaj CPC clip motif uas tuaj yeem dock nrog heparin. Kuj ceeb tias, zoo li LhKNOT thiab lwm cov bona fide knottin proteins, txawm tias Drosophila Drosomycin antifungal peptide qhia CPC clip motif hauv nws cov qauv. Qhov motif no kuj raug txheeb xyuas hauv 41 ntawm 46 ntxiv LhKNOT homologs los ntawm L. heteroatom, L. boulardi, thiab Ganaspis spp. wasps ntse ntawm D. melanogaster.

Cov no hauv silico-raws li kev soj ntsuam qhia tias CPC clip motif, muaj nyob rau hauv ntau lub knottins, yuav muab cov qauv hauv paus rau kev hloov kho lub cev tiv thaiv kab mob lossis kev ua phem. Peb kwv yees txog qhov ua tau los tiv thaiv kab mob lossis kab mob ntsig txog kev ua haujlwm rau tus kab mob LhKNOT. Kev nplua nuj ntawm knottin quav thiab nws concurrence nrog CPC clip motif nyob rau hauv feem ntau ntawm cov tshiab pom knottin peptides los ntawm peb Drosophila parasitoids qhia txog lub luag hauj lwm tseem ceeb uas tseem tsis tau txiav txim siab rau lawv nyob rau hauv parasitoid physiology thiab / los yog nyob rau hauv lub party-parasites. kev sib tham.

2. Cov txiaj ntsig
2.1. Lub LhKNOT ib ntus qhia qhov zoo sib xws rau Kab thiab tsob nroj Antimicrobial Peptides

Kev tshawb nrhiav BLASTp ntawm nr thiab TSA cov ntaub ntawv siv qhov ntev LhKNOT sib lawv liag (60 amino acids) tau pom qee qhov kev txwv tseem ceeb, feem ntau sau tseg raws li "antimicrobial peptides", los ntawm cov nroj tsuag thiab kab. Ob peb cov kab mob fungal kuj tau txheeb xyuas. Tag nrho cov txiaj ntsig tau pom tsawg dua 60 feem pua ​​​​ntawm cov amino acid tus kheej rau LhKNOT. Qhov kev sib tw saum toj kawg nkaus rau LhKNOT hauv nr database yog ib qho tshuaj tua kab mob peptide los ntawm cov kab ntau, Callosobruchus maculates, thaum lub sab saum toj ntaus hauv TSA database yog cov tshuaj tua kab mob peptide los ntawm cov nplej curl mite Aceria Coachella.

Kev txuag yog qhov tseem ceeb tshaj plaws nyob rau hauv cheeb tsam ntawm ib qho kev sib txuas motif txuam nrog LhKNOT, cysteine ​​knotted 'antifungal peptide' kos npe (pfam11410 domain, E=2.8 × 10−3 ). Tom qab khiav PSI-BLAST rau kev tshawb pom ntawm cov chaw taws teeb homlogs, cov kab ke ntxiv tau tshwm sim. PSI-BLAST pom cov txiaj ntsig zoo sib xws nrog cov nroj tsuag dej khov, Mesembryanthemum crystallinum; liab mites, Dinothrombium tinctorium; parasitoid kab mob, Trichogramma pretiosum; nyem kab, Ignelater luminous; kab mob cab, Trichogramma brassicae; Brassicogethes aeneus; tshauv borer, Agrilus planipennis; thiab sawfly, Neodiprion lecontei.

Siv Hidden Markov Models-driven HMMER web server ua lwm txoj hauv kev tshawb xyuas cov chaw taws teeb homologs, peb tau txheeb xyuas tsib qhov fungal hits los ntawm Akanthomyces lecanii, Cordyceps javanica, Cordyceps confragosa, Rosellinia necatrix, thiab Beauveria bassiana.

Kev tshawb fawb ntawm PDB rau qhov zoo sib xws tau txais tsuas yog ob qho txiaj ntsig tseem ceeb, ib qho rau knottin-type antifungal peptide Alo-3 los ntawm kab Acrocinus longimanus thiab qhov thib ob los ntawm disulfide-constrained qhov txhab kho peptide pB1 los ntawm Pereskia bleo. Cov ntsiab lus ntawm tag nrho cov nyob ze thiab tej thaj chaw deb homologs txheeb xyuas siv cov kev sib txawv sib txawv zoo sib xws tau muab sau tseg hauv Cov Lus Ntxiv S1.

Ib qho kev sib raug zoo ntawm LhKNOT mus rau cov qauv paub ntawm cov qauv knottins zoo li cov homologs pom tau pom kev txuag ntawm rau cysteines uas tsim peb tus choj disulfide hauv LhKNOT (Daim duab 1A). Ntawm nws cov N-terminus, LhKNOT muaj qhov ntev, helical ntu kwv yees ua haujlwm raws li lub teeb liab sib lawv liag; sab hauv, nws muaj peb nplooj ntawv beta, raws li qhov xav tau ntawm cov qauv thib ob ntawm knottin peptides (Daim duab 1B).

Tsis tas li ntawd, qhov kev ntsuam xyuas ntawm qhov sib npaug ntawm LhKNOT tau tsom mus rau L. heteroatom thiab L. boulardi cov ntawv sau tseg thiab los ntawm lwm tus Drosophila parasitoid, Ganaspis spp. (saib Cov Txheej Txheem), nthuav tawm ntau qhov sib xws. Rau L. heterotoma (Lh14), peb tau txheeb xyuas 16 ntxiv Lh14 knottin proteins (Supplementary Table S2).
Rau L. boulardi, peb txheeb xyuas 22 knottin proteins (Sab Ntxiv Table S3). Rau LhKNOT homologs tau zoo sib xws los ntawm Ganaspis hookeri (G1) thiab ob LhKNOT homologs tau pom nyob rau hauv cov protein kwv yees ua los ntawm G. brasiliensis (VA) genome (Table Ntxiv S4). Daim duab 2 qhia txog kev txuag ntawm cov kab mob parasiteid knottin nrog LhKNOT. Yog li, tsawg kawg yog ob peb tus kab mob Drosophila uas tau kuaj pom hauv qhov kev tshawb fawb no tshwm sim los encode ntau knottin proteins. Ntxiv xws li knottin peptides tej zaum yuav encoded los ntawm cov wasps, thiab systematic proteomic/bioinformatic mus kom ze yuav tsum tau kom tau ib daim ntawv qhia. Lub luag haujlwm ntawm cov pob txha pob txha hauv cov kab mob physiology tseem tsis paub.

Daim duab 1. (A) Ntau qhov sib txawv ntawm LhKNOT (sab saum toj) thiab homologs hauv tsev neeg knottin (sab saum toj-rau-hauv qab): Mesembryanthemum crystallinum (cog; tshuaj tua kab mob; NCBI ID: AAC19399), Aceria tosichella (mite; antimicrobial; NCBI ID :MDE48292.1), Callosobruchus maculatus (beetle; tsis paub; NCBI ID:VEN35376.1), Dinothrombium tinctorium (mite; tsis paub; NCBI ID:RWS16713.1), Trichogramma pretiosum (Alo{{10}} xws li; parasitoid wasp; antimicrobial; NCBI ID: XP_014233229.1), Ignelater luminosus (beetle; tsis paub; NCBI ID: KAF2884324.1), Trichogramma brassicae (parasitoid wasp; tsis paub; NCBI ID: CAB{{27 }}{{30}}31014.1), Brassicogethes aeneus (beetle; tsis paub; NCBI ID: CAH0563829.1), Agrilus planipennis (ash borer; antimicrobial; NCBI ID : XP_018321119.1), Neodiprion lecontei (sawfly; tsis paub; NCBI ID: XP_015517007.2), Akanthomyces lecanii (fungus; antifungal; Uniprot ID: A0A168C5L6), Cordyceps javanica (fungus; antifungal) ; Uniprot ID: A0A545VVU1), Cordyceps confragosa (fungus; tsis paub; Uniprot ID: A0A179IJT7), Rosellinia necatrix (fungus; tsis paub; Uniprot ID: A0A1S8A723), Beauveria bassiana (fungus; tsis paub; Uniprot ID: A0A0A2VUF6), Acrocinus longimanus (Alo-3; beetle; antifungal; PDB ID: 1Q3J), thiab Pereskia bleo (pB1; ID: usB1; 5 XAB). Cov residues zoo ib yam yog qhov tseem ceeb hauv cov xim liab, thaum cov tshuaj lom neeg zoo sib xws tau pom hauv cov xim daj. Cov xub dub qhia txog cov cysteine ​​residues uas yog lub luag haujlwm rau lub pob txha pob txha pob txha, thiab tus choj disulfide txuas ntawm peb tus choj disulfide yog cim nrog neon ntsuab tooj.

X thiab XX qhia txog qhov sib txawv ntawm cov seem ntawm N- thiab C-terminus, feem. Cov txheej txheem thib ob ntawm cov qauv LhKNOT tau qhia rau saum. thiab T sawv cev -strand, thiab -tig, feem. (B) Qhov thib ob qauv kwv yees rau LhKNOT, uas qhia txog qhov ntev helical ntu (lub tog raj kheej liab; teeb liab peptide) ua raws li peb beta strands (daj xub). Cov qauv theem nrab no ua raws li kos npe canonical Knottin [36]. (C) Ib tug qauv ntawm cov qauv knottin folds los ntawm KNOTTIN database [36] (tso cai rov tsim dua daim duab no muab los ntawm Dr. Jean-Christophe Gelly, INSERM, Lub Kaum Hli 2019). Ob qhov txuas txuas hniav disulfide (ntsuab) tsim ib lub voj voog macro, thaum lub thib peb (xiav) rub los ntawm qhov chaw muab cov protein nws lub npe hu ua knotted topology. (D) Cov qauv kev kwv yees tertiary ntawm LhKNOT, uas qhia cov pob txha topology thiab kev txuag cysteine ​​motif, cysteine ​​residues tsim cov txuas hniav disulfide hauv cov qauv kev txuag pom thoob plaws hauv tsev neeg knottin (C1 -C4 thiab C2 -C5 daim ntawv macro-cycle, C3 -C6 rub los ntawm qhov chaw).

Daim duab 2. (A) Ntau qhov sib txuas ua ke ntawm LhKNOT (sab saum toj) thiab homologs hauv Lh14. (B) Ntau qhov sib txuas ua ke ntawm LhKNOT (sab saum toj) thiab homologs hauv L. boulardi. Lub X cim nyob rau hauv lub thawv ntsuab sib raug rau cov kab lus hauv qab no hauv ob lub Lb17 ib ntus: Lb: GISX01151661.1 X=SQAPPPTPEPFHPPGTPP; Lb: GISX01151653.1 X=SQLTSPRSTFPPTGSTIPSIITT. (C) Ntau qhov sib txuas ua ke ntawm LhKNOT (sab saum toj) thiab homologs hauv Ganaspis spp. Cov residues zoo ib yam yog qhov tseem ceeb hauv cov xim liab, thaum cov tshuaj lom neeg zoo sib xws tau pom hauv cov xim daj.

Cov xub dub qhia txog cov cysteine ​​residues uas yog lub luag haujlwm rau lub pob txha pob txha pob txha, thiab tus choj disulfide txuas ntawm peb tus choj disulfide yog cim nrog neon ntsuab tooj. Cov txheej txheem thib ob ntawm cov qauv LhKNOT tau qhia rau saum. thiab T sawv cev -strand, thiab -tig, feem. Grey hnub qub tau ntxiv rau saum cov blocks ntawm cov kab ke, cov seem saum toj no sawv cev rau cov khoom seem ua qauv nrog lwm qhov sib txawv.

2.2. Tus Qauv Peb-Dim ntawm LhKNOT

Cov txheej txheem zoo ib yam ntawm knottin peptide yog txhais los ntawm 6 cysteine ​​koom nrog hauv peb daim ntawv cog lus disulfide los ua ib qho cysteine-knotted peb-stranded antiparallel beta-daim ntawv [25]. Ib qho tseem ceeb ntawm qhov quav no yog kev sib txuas ntawm cov cysteine ​​residues los tsim cov txuas hniav disulfide thiab tus choj disulfide ntawm cysteine ​​3 thiab 6 mus rau disulfides 1-4 thiab 2-5 los ua ib qho tshwj xeeb disulfide los ntawm disulfide knot (Daim duab 1A, C. ).

Qhov no disulfide, los ntawm ib tug disulfide knot feature, distinguishes lub knottin quav los ntawm lwm yam cysteine ​​knot proteins. Lwm yam tshaj li cov cysteine ​​khaws cia, qhov seem ntawm cov amino acid ib ntus tuaj yeem sib txawv heev, thiab yog li ntawd, tsuas yog qee tus lej ntawm knottins raug txheeb xyuas los ntawm cov txheej txheem zoo sib xws rau LhKNOT (saib tshooj dhau los thiab Cov Lus Qhia Ntxiv S1).

Fold recognition algorithms thiab template-based modeling approaches, ntawm qhov tod tes, tau muab ntau yam sib txawv knottins raws li cov neeg sib tw tsim nyog rau kev ua qauv LhKNOT. Ntawm cov qauv LhKNOT ntau yam tsim tawm, tus qauv zoo tshaj plaws yog ntau tus qauv-raws li qauv ntawm LhKNOT tsim siv qhov kev pab cuam HHpred [37] nrog cov txheej txheem tswj tau los ntawm peb cov qauv saum toj kawg nkaus, Alo-3 (PDB: 1Q3J) [ 30], omega-agatoxin-IVA (PDB: 1IVA) [28], thiab PAFP-S (PDB: 1DKC) [25]. Kev paub txog kev siv hluav taws xob siv hluav taws xob suav nrog ProSA-web [38] rau cov qauv xaiv no tau pom muaj zog qis thiab z-score ntawm −4.53, uas yog piv rau cov qauv daws teeb meem.

Verify3D [39] tau ntsuas qhov ntau tus qauv-raws li tus qauv nrog tus qhab nia dhau (100.00 feem pua ​​​​ntawm cov seem hauv tus qauv muaj qhov nruab nrab 3D-1 D tau qhab nia ntau dua lossis sib npaug rau 0.2). Lub VoroMQA [40] qhab nia ntawm 0.325 yog qhov zoo li qhov kev xav ntawm qhov me me ntawm peptide thiab cov qhab nia ntawm cov qauv nyob rau tib qhov ntau (Cov duab ntxiv S1).

Cov qauv siv ntau yam no ua rau cov qauv knottin folds nrog peb qhov sib npaug ntawm cov beta-strands, txwv tsis pub cov txuas hniav disulfide thiab kev cia siab txuas ntawm cov txuas hniav disulfide (Daim duab 1D). KNOTER3D, ib lub cuab yeej hauv KNOTTIN Database [36] corroborated tias cov qauv no muaj ib tug bona fide knottin quav thiab paub tseeb tias yav tas los txheeb xyuas disulfide bonding qauv hauv daim duab 1D.

2.3. LhKNOT Qhia txog qhov muaj CPC Clip, muaj peev xwm cuam tshuam nrog Heparin thiab lwm yam GAGs

Txhawm rau tshawb nrhiav LhKNOT muaj peev xwm ua haujlwm, cov qauv-kev ua haujlwm sib raug zoo ntawm cov qauv zoo sib xws ntawm LhKNOT tau tshawb xyuas. Ib qho knottin protein los ntawm cactus Pereskia bleo (PDB: 5XBD) [26] muaj cov khoom siv heparin-binding motif hu ua CPC clip. Cov qauv qauv no tsim cov duab geometrically constrained clip ntawm ob cationic thiab ib tug polar residues uas cuam tshuam nrog heparin los ntawm ntsev txuas hniav thiab hydrogen bonds clamping nws nyob rau hauv txoj hauj lwm nyob rau hauv qhov zoo them nto ntawm lub clip [41].

Ib tug qauv superposition ntawm cactus knottin thiab LhKNOT tau pom tias ob lub qauv zoo sib xws, nrog rau tag nrho qhov sib txawv ntawm tsawg dua 2Å. Docking tsom xam ntawm LhKNOT nrog heparin, siv ClusPro cov kev txwv tshwj xeeb rau heparin raws li lub ligand, qhia tias rau LhKNOT, cov polar sib cuam tshuam haum rau CPC clip motif tshwm sim nyob rau hauv seem R1-S3-R14 (Daim duab 3A) .

Kev ntsuas qhov deb (ob qho tib si ntawm alpha-carbons thiab cov chaw nruab nrab ntawm cov khoom seem ntawm cov khoom seem) sib txawv ntawm cov CPC clip los ntawm tsawg dua 1Å, tawm tswv yim txog qhov muaj qhov tso tawm CPC clip hauv LhKNOT (Daim duab 3B). Qhov saum npoo electrostatics ntawm tus qauv LhKNOT kuj pom tias muaj qhov xav tau cationic binding hnab tshos tsim los ntawm CPC clip [41,42] (Daim duab 3C).

Txhawm rau soj ntsuam CPC clip lub peev xwm los cuam tshuam nrog lwm cov GAGs, kev tshuaj xyuas txuas ntxiv tau ua nrog LhKNOT thiab keratin sulfate (PDB ID: 1KES) thiab nrog LhKNOT thiab hyaluronic acid (PDB ID: 1HYA). Nyob rau hauv ob qho tib si, qhov zoo nqi khi nto tsim los ntawm CPC clip yog khi rau qhov tsis zoo nqi molecules. Liglots tsim los ntawm PDBSum tau lees paub tias muaj kev koom tes ntawm GAGs nrog CPC-forming residues (Cov duab ntxiv S2 thiab S3).

2.4. Lwm Cov Qauv Homologs ntawm LhKNOT Tseem Muaj CPC Clip Motif

Lub xub ntiag ntawm CPC clip nyob rau hauv ob qho tib si LhKNOT thiab nws cov qauv homolog los ntawm Pereskia bleo (pB1) qhia tau hais tias muaj peev xwm ntawm ib tug dav kev txuag ntawm CPC clip nyob rau hauv lub knottin tsev neeg. Kev tshuaj xyuas ntawm 21 cov qauv zoo sib xws ntawm LhKNOT nrog kev sim daws PDBs hauv KNOTTIN cov ntaub ntawv qhia tias, hauv txhua kis, cov peptides muaj CPC clip motif. Rau feem ntau peptides (piv txwv li, T. tridentatus), cov motif tsis deviated tsawg tshaj li 1Å los ntawm cov tub ntxhais knottin quav, thaum rau lwm tus neeg (xws li, lub offensive knottins), lub deviation yog mus txog rau ib tug margin ntawm 3Å (Supplementary Table S5). Ib yam li ntawd, cov qauv knottin cov proteins ntawm cov homologs pom tau tias yuav luag txhua tus ntawm lawv muaj CPC clip.

Kaum rau qhov sib txuas ntxiv los ntawm L. heteroatom, 22 ntu ntawm L. boulardi, thiab 8 ntu ntawm Ganaspis spp., uas tsis paub cov qauv 3D, tau ua qauv thiab tshuaj xyuas rau lub xub ntiag ntawm CPC clip motif. Thaum 13 ntawm 16 putative L. heteroatom knottins thiab 20 ntawm 22 putative L. boulardi knottins tau nthuav tawm CPC clip motif, tag nrho Ganaspis sequences muaj CPC clip (Table Ntxiv S5). Interestingly, xya homologs uas tsis tau nthuav tawm CPC clip motif yog (a) kab paj ntoos kab ke (B. aeneus (CAH0563829.1), (b) fungal sequence R. necatrix (A0A1S8A723), thiab (c) peb. L. heterotoma knottins 4,5,6 thiab L. boulardi knottins 3 thiab 20 (Cov Lus Qhia Ntxiv S5).

Kev tshawb fawb ntawm knottins hauv KNOTTIN database, tsis cuam tshuam rau LhKNOT, tab sis tus neeg sawv cev ntawm ntau cov se (cone snail, horseshoe crab, insect, plant, scorpion, and spider), qhia CPC clip motif. Nyob rau hauv txhua rooj plaub, qhov saum npoo ntawm electrostatic profile ntawm CPC clip motif yog qhov zoo them, zoo li, los pab txhawb kev sib cuam tshuam nrog cov tsis zoo them heparin (Daim duab 4). Txawm li cas los xij, zoo ib yam li cov kab, fungal, thiab wasp knottins tau piav qhia saum toj no, CPC clip tsis tau txheeb xyuas hauv knottins los ntawm cov nroj tsuag cyclotide thiab sponges (Table Ntxiv S5).

Daim duab 4. Heparin-docking tau thiab nto electrostatics ntawm (A) antifungal peptide ntawm Phytolacca americana (PFAP-s; cog; PDB: 1DKC), (B) calcium channel xaiv omega-agatoxin ntawm Agelenopsis aperta (Omega-agatoxin IV-A ; kab laug sab; PDB: 1IVA), (C) antifungal peptide ntawm Acrocinus longimanus (Alo-3; beetle; PDB: 1Q3J), (D) qhov txhab kho peptide ntawm Pereskia bleo (pB1; cactus; PDB: 5XBD) , (E) LhKNOT, (F) antifungal peptide "Drosomycin" ntawm Drosophila spp. (Drosomycin; ya; PDB: 1MYN), (G) Conotoxin GS (Conotoxin GS; cone snails; PDB: 1AG7), (H) antimicrobial peptide Tachystatin A cais los ntawm Horseshoe crabs (Tachystatin A; horseshoe crabs; PDB: 1CIX) thiab (I) Chlorotoxin, me me scorpion toxin ntawm Leiurus quinquestriatus (Chlorotoxin; scorpion; PDB: 1CHL) qhia cov cationic CPC clip motif. Cov duab taw qhia kom pom qhov zoo tshaj plaws CPC clip qauv. Qhov saum npoo electrostatic muaj peev xwm yog xim-qib los ntawm −4 kT / e (liab) rau ntxiv rau 4 kT / e (xiav).

2.5. Hauv Silico Mutational Analysis ntawm LhKNOT CPC Clip nthuav tawm qhov CPC Thib Ob

Cov qauv ntawm peb hauv silico LhKNOT mutants (R1A, S3A, R14A) tau tshawb xyuas los sib piv kev sib txuas ntawm cov tsiaj qus thiab mutant proteins. Nyob rau hauv rooj plaub ntawm R14A mutant, heparin tau pom tias muaj kev cuam tshuam nrog CPC txuas ntxiv nrog rau R1, S3, thiab R30 residues es tsis txhob R1, S3, thiab R14 motif nyob rau hauv cov tsiaj qus (Ntxiv daim duab S4). Muab ob npaug los yog triple mutants (R14A-R1A; R14A-S3A-R1A), txawm li cas los xij, tsis tau qhia txog kev cuam tshuam nrog ib qho ntawm thawj lossis theem nrab CPC clip motif residues (Cov duab ntxiv S4C, D).

Cov hauv silico mutants nrog R1A kev hloov pauv (kev cuam tshuam ob qho tib si thawj thiab theem nrab CPC clip motifs), txawm li cas los xij, tau qhia tias heparin koom nrog R29 thiab R30, raws li lawv muab qhov seem cationic nkaus xwb (Cov Duab Ntxiv S4C, D). Interestingly, lub ligand nyiam mus koom nrog CPC clip motif, ob qho tib si nyob rau hauv cov ntaub ntawv ntawm thawj thiab theem nrab clips, tshaj no ntxiv cationic nto thaum lub motif muaj, tawm tswv yim txog kev sib cuam tshuam ntawm CPC clip yuav tso cai rau ib tug ntau ruaj khov koom haum. kev sib cuam tshuam ntawm electrostatic tsis tshwj xeeb.

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