Hauv Silico Kev Ntsuam Xyuas Ntawm Drosophila Parasitoid Venom Peptide Qhia Txog Kev Tshaj Tawm Ntawm Cation-Polar-Cation Clip Motif hauv Knottin Proteins Part 2
Aug 04, 2023
2.6. CPC Clip tseem muaj nyob rau hauv Antimicrobial Peptide Drosomycin
Tsis zoo li LhKNOT, cov qauv ntawm Drosomycin muaj cov quav sib txawv nrog cov alpha-helix thiab ib daim ntawv sib tw peb-stranded beta-daim ntawv, uas yog ruaj khov los ntawm peb cov txuas hniav disulfide [43]. Cov qauv qauv no, hu ua "cysteine stabilized -motif", kuj pom muaj nyob rau hauv lwm cov tshuaj tiv thaiv kab mob xws li cov kab mob tua kab mob A, scorpion toxins, cog thionins, thiab tshuaj tua kab mob nroj tsuag defensins [43,44].
-helix yog polypeptide uas muaj kev ua haujlwm lom neeg siab, uas tuaj yeem ua rau tib neeg lub cev tiv thaiv kab mob thiab txhim kho tib neeg kev tiv thaiv, yog li tiv thaiv thiab kho ntau yam kab mob. Cov kev tshawb fawb tau pom tias -helix muaj cov txiaj ntsig ntawm kev txhim kho kev tiv thaiv kab mob, tuaj yeem txhim kho phagocytic muaj peev xwm ntawm macrophages thiab kev ua haujlwm ntawm T hlwb, txhim kho lub cev tiv thaiv kab mob sab nraud, txo lub cev rhiab heev rau qhov chaw tsis zoo, thiab txhawb kev noj qab haus huv.
Kev tiv thaiv kab mob tseem ceeb heev rau tib neeg kev noj qab haus huv. Nws tuaj yeem pab peb tiv taus ntau yam kab mob, ua kom lub cev muaj zog, thiab txhim kho kev tiv thaiv kab mob. Txawm li cas los xij, tib neeg nyob hauv ib puag ncig uas muaj ntau yam cuam tshuam rau cov kab mob thiab ib puag ncig muaj kuab paug, thiab kev tiv thaiv tau yooj yim cuam tshuam. Yog li ntawd, txhim kho kev tiv thaiv kab mob yog qhov tsim nyog rau kev tswj hwm tib neeg kev noj qab haus huv.
Raws li ib tug natural active peptide, -helix tsis tau tsuas yog tswj lub cev tiv thaiv kab mob thiab txhim kho kev tiv thaiv kab mob, tab sis kuj plays lub luag hauj lwm zoo ntawm kev tiv thaiv kab mob plawv thiab metabolic ntshawv siab, txhim kho daim siab ua haujlwm, txo cov ntshav lipids, thiab ntshav qab zib, thiab txhim kho lub cev muaj zog thiab tiv thaiv. - qaug zog muaj peev xwm. Yog li ntawd, -helix tau siv dav hauv kev lag luam tshuaj, khoom siv kho mob, thiab tshuaj pleev ib ce, tau ua tiav cov txiaj ntsig zoo kawg, thiab tau txais tos txais tos.
Hauv luv luv, -helix yog ze ze rau tib neeg kev tiv thaiv, nws tuaj yeem txhawb kev tiv thaiv tib neeg thiab ua lub luag haujlwm tseem ceeb hauv kev ua kom tib neeg noj qab haus huv thiab kev ua neej ntev. Nyob rau hauv lub neej yav tom ntej thiab kev ua hauj lwm, peb yuav tsum nquag txhim kho kev tiv thaiv kab mob, txhim kho kev ua neej nyob, kev tawm dag zog thiab kev noj zaub mov kom raug, xav txog kev siv -helix los txhim kho kev tiv thaiv, thiab ua kom muaj kev sib tw thiab cov cib fim yav tom ntej. 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 polysaccharides hauv Cistanche tuaj yeem tswj kev tiv thaiv kab mob ntawm tib neeg lub cev tiv thaiv kab mob, txhim kho kev ntxhov siab ntawm lub cev tiv thaiv kab mob, thiab txhim kho cov kab mob bactericidal ntawm lub cev tiv thaiv kab mob.
Nyem cistanche deserticola ntxiv
Muab lub npe hu ua antimicrobial muaj nuj nqi ntawm Drosomycin thiab nws cov cuam tshuam "cysteine stabilized -motif" [43,44], peb tau tshuaj xyuas qhov muaj peev xwm ntawm cov hauv paus rau kev siv tshuaj tua kab mob thiab cov qauv sib koom-kev sib raug zoo nrog LhKNOT kom nkag siab zoo dua qhov tseem ceeb ntawm cov qauv no. nyob rau hauv LhKNOT hauv cov ntsiab lus ntawm kev tiv thaiv tus tswv.
Txawm hais tias muaj qhov sib txawv me ntsis, Drosomycin overlays nyob rau hauv RMSD ntawm 2.2Å ntawm LhKNOT thiab pB1 (Daim duab 5A). Thaum yav dhau los CPC clip tsom xam tau txuas ntxiv mus rau Drosomycin, cov txiaj ntsig tau qhia tias muaj CPC clip motif hauv Drosomycin thiab. CPC clip motif nyob rau hauv Drosomycin (R6-S4-K38) ntog nyob rau hauv ib tug 2Å sib txawv ntawm cov motif tsis (Daim duab 5B, Supplementary Table S5).
Tsis tas li ntawd, deg electrostatics ntawm Drosomycin tau lees paub qhov kev tso tawm CPC clip tsim ntawm cationic binding hnab tshos raws li tau pom zoo rau LhKNOT thiab lwm yam knottins (Daim duab 5C, Cov lus ntxiv S5).
Cov txiaj ntsig no qhia tias tej zaum yuav muaj qhov zoo sib xws hauv hom kev ua ntawm Drosomycin thiab LhKNOT thiab tias biochemistry embedded nyob rau hauv cov qauv motif no yuav tsav antimicrobial muaj nuj nqi nyob rau hauv ob qho tib si peptides.
Daim duab 5. (A) Superposition ntawm LhKNOT (xiav), qhov txhab kho peptide ntawm Pereskia bleo (tan; pB1; cactus; PDB: 5XBD) thiab Drosomycin (ntsuab; Drosomycin; ya; PDB: 1MYN). (B) Qhov deb tau ntsuas hauv PyMOL nruab nrab ntawm cov chaw nruab nrab ntawm cov khoom seem uas koom nrog (grey pseudo-atoms) hauv kev sib cuam tshuam ntawm Drosomycin-heparin. Qhov kev ncua deb poob hauv 2Å ntawm tus nqi ntau ntawm CPC clip motif. (C) Nto electrostatics ntawm Drosomycin (fly; PDB: 1MYN), uas qhia qhov zoo them nqi khi nto tsim los ntawm CPC clip qhov twg heparin yog docked. Qhov saum npoo electrostatic muaj peev xwm yog xim-qib los ntawm −4 kT / e (liab) rau ntxiv rau 4 kT / e (xiav).
3. Kev Sib Tham thiab Kev Sib Tham
Kev ua haujlwm biochemical ntawm cov protein tau yooj yim los ntawm lawv cov qauv tag nrho, uas tau tshaj tawm 3-10 npaug ntau dua kev txuag dua li lawv cov qauv tseem ceeb [45]. Yog li ntawd, kev sib txuas ua ke feem ntau poob qis hauv kev tshawb xyuas cov peev xwm ua haujlwm ntawm cov proteins xws li knottins, qhov twg cov qauv sib txawv tuaj yeem ua rau tsis pom kev sib koom ua ke-kev sib raug zoo.
Hauv cov txheej txheem silico pab txhawb kev tsom xam dav dav ntawm cov qauv muaj peev xwm-kev ua haujlwm sib raug zoo rau cov kev nthuav dav zoo li no [46–50]. Peb cov ncauj lus kom ntxaws tsom xam ntawm LhKNOT qauv thiab nws cov qauv homologs txhawb lub tswv yim tias cov kab mob parasite no muaj cov cim ntawm knottin peptide nrog kev ua haujlwm hauv kev tiv thaiv tus tswv: (a) qhov sib tw ze tshaj plaws rau LhKNOT yog cov tshuaj tua kab mob peptides; (b) LhKNOT's Pfam kos npe piav qhia nws li cysteine knotted "antifungal peptide"; thiab (c) CPC clip yog tam sim no.
Kev tshuaj xyuas ntawm knottin qhov txhab-kho peptide login pB1 los ntawm nplooj ntoo cactus Pereskia bleo pib qhia txog qhov muaj CPC clip, kev txuag kev kos npe ntawm heparin-binding proteins [42]. Nws muaj kev tiv thaiv- thiab kev taw qhia txog kev ua haujlwm hauv kev kho mob thiab mob qog noj ntshav thiab siv los ua cov tshuaj tiv thaiv coagulant [51].
GAGs muaj nyob rau ntawm cov tsiaj ntawm tes thiab tau dhau los ua lub hom phiaj nrov ntawm cov kev tshawb fawb xws li kev kho mob mus rau tshuaj pleev ib ce [52]. CPC clip motif ntawm heparin-binding proteins yog suav tias yog qhov tsawg kawg nkaus xav tau motif rau heparin-binding affinity thiab yog txhais los ntawm kev sib raug zoo spatial ntawm peb seem, ob cationic (Arg / Lys) thiab ib tug polar (Asn, Gln, Ser, Thr, Tyr).
Qhov CPC clip motif no yog txhais los ntawm qhov kev ncua deb ntawm ob qho tib si cov pa roj carbon monoxide residues nrog rau qhov kev ncua deb ntawm qhov chaw ntawm qhov loj ntawm cov khoom seem. Heparin-binding proteins paub tias muaj kev cuam tshuam nrog lwm cov substrates tsis zoo, tshwj xeeb tshaj yog cov kab mob ntawm cov kab mob microbes [41].
Raws li qhov tshwm sim, heparin-binding proteins tuaj yeem pom cov tshuaj tiv thaiv kab mob rau ntau qib [53]. Txawm hais tias cov txheej txheem meej ntawm cov tshuaj tiv thaiv kab mob no tsis tau txhais tau zoo, nws tau pom zoo tias CPC clip tuaj yeem pab txhawb cov protein-microbe kev sib cuam tshuam [41]. Lub luag haujlwm tshiab muaj peev xwm ntawm heparin-binding tau tshwm sim, xws li hauv cov peev txheej kho mob rau amyloidogenesis [54], yog li kev nkag siab txog cov qauv txiav txim siab ntawm CPC clip nyob rau hauv ntau cov proteins yuav yog qhov tseem ceeb rau delineating lub mechanism ntawm kev sib cuam tshuam thiab nws cuam tshuam.
Cov lus qhia rau qhov ua tau los tiv thaiv kab mob rau LhKNOT los ntawm peb qhov kev tshawb pom tias nws cov inhibitory cysteine knot thiab cov ntawm lwm cov knottins kuaj ntawm no muaj CPC clip intrinsic rau lawv cov quav (Sab Ntxiv Table S5).
Nws tau raug sau tseg tias cov qauv thiab biophysical yuav tsum muaj rau heparin-binding proteins 'muaj peev xwm cuam tshuam nrog heparin yog strikingly zoo ib yam li cov qauv ntawm ntau yam paub AMPs [53]. Yog li, CPC clip motif tuaj yeem haum rau cov qauv rau ob qho kev cai no [41].
Peb qhov kev tshawb pom ntawm CPC clip motif no tsis yog nyob rau hauv LhKNOT nkaus xwb tab sis kuj nyob rau hauv 75 lwm yam los yog tsis muaj feem cuam tshuam nrog knottin proteins (Sab Ntxiv Table S5) qhia tias qhov CPC clip motif no yuav yog ib qho kev txuag ntawm cov proteins knottin.
Qhov xwm txheej tshwm sim ntawm CPC clip motif nyob rau hauv lub cysteine knot qauv nyob rau hauv ntau yam proteins qhia tias CPC clip motif nws tus kheej yuav underlie lub antimicrobial muaj nuj nqi. Txawm li cas los xij, vim tias cov qauv tsim qauv no kuj tau txheeb xyuas hauv kev ua txhaum kev sib tsoo (Sab Ntxiv Table S5), nws kuj tseem tuaj yeem ua haujlwm ntau dua los ntawm qhov kev sib cuam tshuam ntau dua ntawm CPC clip motif.
Qhov kev tshawb pom ntawm putative CPC clip motif nyob rau hauv Drosomycin yog intriguing, raws li nws tso lub teeb ntawm lub peev xwm ntawm kev txiav txim ntawm no antifungal peptide thiab txhawb ib tug ua tau antimicrobial muaj nuj nqi rau CPC clip motif nws tus kheej. Cov kev tshawb fawb yav dhau los tau qhia qhov tseem ceeb ntawm Drosomycin cov residues R6 thiab K38, uas yog ib feem ntawm nws CPC clip motif (R6-S4-K38), rau nws cov peev xwm tiv thaiv kab mob; Kev hloov pauv ntawm cov khoom seem no txo qis kev ua haujlwm ntawm cov kab mob fungal [55]. Peb cov qauv kev soj ntsuam ntawm tus kab mob LhKNOT thiab nws qhov zoo sib xws nrog Drosophila Drosomycin muaj peev xwm nkag siab txog L. heterotoma lub peev xwm los nthuav dav txoj hauv kev TollNF-κB hauv D. melanogaster thiab tseem ua tiav ntawm kev tiv thaiv kab mob.
Thaum tsis muaj txoj hauv kev tiv thaiv kab mob ua haujlwm, thiab yog li ntawd, tsis muaj Drosomycin ntau lawm, Drosophila larvae feem ntau tuaj yeem ua rau cov kab mob kis tau zoo [11,13,56]. LhKNOT cov tshuaj tiv thaiv kab mob tuaj yeem tiv thaiv tus tswv tsev los ntawm cov kab mob kis tau zoo hauv cov kab mob kis kab mob, tiv thaiv kab mob tiv thaiv kab mob thiab tsim cov pupae. Xwb, zoo ib yam li Drosomycin, LhKNOT tuaj yeem ua rau hemocyte apoptosis [57]. L. heteroatom EVs nkag mus rau hemocytes tom qab kis kab mob [58], thiab lawv cov dej num hauv EVs cuam tshuam nrog encapsulation [19,21].
CPC clip tau pom tias muaj kev sib raug zoo nrog GAGs [41], uas tseem muaj nyob rau hauv kab nrog rau Drosophila [59]. Heparin sulfate GAGs nyob rau hauv yoov pab raws li kev loj hlob receptors thiab koom nrog tsim thiab tswj morphogenic gradients [60]. CPC clip kuj tau pom tias koom nrog cov lipopolysaccharides (LPS) [41]. Cov kab mob LPS, yog qhov tseem ceeb ntawm cov kab mob Gram-negative, yog cov muaj zog heev stimulators ntawm lub cev tiv thaiv kab mob hauv ntau hom eukaryotic nrog rau kab [61]. GAGs nyob rau hauv Drosophila kuj tswj kev khi ntawm C protein, ib tug kab mob determinant ntawm pab pawg B streptococcus [62].
Yog li, nws muaj peev xwm hais tias CPC clip uas muaj LhKNOT thiab Drosomycin (thiab tejzaum nws muaj lwm yam yoov AMPs, xws li Defensin [63]) tuaj yeem hloov kho lub cev tiv thaiv kab mob (thiab/lossis khi rau cov kab mob virulence proteins), zoo sib xws rau cov kab mob pathogen/metazoan. parasite tshwm sim. Cov kev tshawb fawb yav dhau los tau pom tias peb homologous knottins (Alo-1, 2, 3) los ntawm harlequin beetle Acrocinus longimanus yog nquag tawm tsam cov poov xab, Candida glabrata [30]. Tsis tas li ntawd, qhov pore-forming candidacidal peptide, Psacotheasin, tawm tsam Candida albicans [64]. Thaum kawg, cov nroj tsuag kab mob xeb fungus, Melampsora lyrics-nrov, tsim ib knottin peptide raws li nws cov neeg sib tw effector [65].
Peb qhov kev tshawb pom tias ntau yam putative knottin peptides tuaj yeem zais los ntawm cov neeg laus cov kab mob cab ntawm Drosophila yog qhov xav tsis thoob thiab xav tsis thoob. Txawm hais tias lawv cov dej num tsis paub, qhov muaj peev xwm muaj nyob ntawm tsev neeg knottin noob qhia qee qhov kev rov ua dua hauv lawv cov dej num uas yuav txuas nrog cov kab mob hauv lub neej keeb kwm. Kev tshawb nrhiav ntawm knottin cov txheej txheem quav hauv qhov tsis sib xws thiab tseem-to-tau-nrhiav knottins yuav ua pov thawj tias muaj txiaj ntsig. Peb txoj haujlwm muab cov npe luv luv ntawm cov khoom seem tseem ceeb rau kev ua haujlwm ntawm lub cev rau kev tshawb nrhiav Knottin tsev neeg protein. Cov ntaub ntawv zoo li no tuaj yeem muab tso rau hauv kev tshawb nrhiav tshuaj muaj txiaj ntsig thiab tsim qauv rau cov kab mob sib kis thiab lwm yam kev kho mob.
4. Cov txheej txheem
4.1. Kev soj ntsuam ib ntus
Kev tshuaj ntsuam thawj zaug ntawm NCBI non-redundant protein sequence (nr) database (https://www.ncbi.nlm.nih.gov/protein; nkag rau 4 Plaub Hlis 2018), Transcriptome Shotgun Assembly (TSA) sequences database (https: //www.ncbi.nlm.nih.gov/genbank/tsa; accessed on 16 April 2018) [66], and the Protein Data Bank (PDB; https://www.rcsb.org; accessed on 5 July 2018) [67] nrog rau tag nrho-ntev amino acid ib ntus ntawm L. heteroatom knottin peptide (GAJC01011813.1) siv NCBI BLAST (BLASTp; https://blast.ncbi.nlm.nih.gov/ Blast. cgi; nkag mus rau 16 Lub Plaub Hlis 2018) [68] tau siv los khaws cov protein sib txuas hauv lwm cov kab mob. Cov chaw taws teeb homlogs tau tshawb nrhiav siv Txoj Haujlwm-Specific Iterative BLAST (PSI-BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi; nkag rau 4 Plaub Hlis 2022) [69], thiab Hidden Markov Models (HMMER; https://www.ebi.ac.uk/Tools/hmmer; nkag rau 6 Plaub Hlis Ntuj 2022) [70,71]. Kev tshawb nrhiav BLAST tau hloov kho rau lo lus loj 2, thiab cov qhab nia matrix BLOSUM45, thaum lub sijhawm tsis raug siv rau HMMER.
Txhawm rau txheeb xyuas cov homologs ntawm knottin hauv xaiv Leptopilina thiab Ganaspis spp. tsis tuaj yeem nkag mus rau hauv cov ntaub ntawv protein, cov kev tshawb fawb tsom siv cov lus txhais BLASTs tiv thaiv cov ntaub ntawv genomic thiab transcriptomic tau ua haujlwm. Txhawm rau txheeb xyuas L. heteroatom knottins ntxiv, L. heteroatom poj niam plab plab transcriptome GAJC [72] thiab tag nrho-lub cev transcriptomes los ntawm av GHUQ (GenBank Accession: GHUP00000000; TSA: Leptopilina heteroatom strain Lh14 thiab shotunt) Chaw GHUP (GenBank Accession: GHUQ00000000; TSA: Leptopilina heteroatom strain Lh14, transcriptome shotgun) tau tshawb fawb siv tblastn (e-tus nqi pib teem rau 1, BLOSUM62, tsis tshua muaj lim lim tau qhib, nug txog kev pab cuam lim lim teem rau 20 feem pua. ) tawm tsam LHKNOT.
Cov ntawv teev lus hauv qab no tau tshawb nrhiav zoo ib yam los txheeb xyuas L. boulardi knottins: L. boulardi poj niam plab plab transcriptome GAJA [72] thiab tag nrho-lub cev transcriptomes los ntawm av GITC (GenBank Accession: GITC00000000; TSA: Leptopilina boulardi, transcriptome shotgun), Space GISX (GenBank Accession: GISX00000000}; TSA: Leptopilina boulardi strain Lb17, transcriptome shotgun), and GGGI00000000 (poj niam plab / taub hau) [73]. Tsis tas li ntawd, tsom mus nrhiav Ganaspis spp. homologs tau ua tiav. For G. hookeri, the GAIW00000000 TSA: Ganaspis spp.
G1 dataset [74] tau tshawb nrhiav (tblastn, e-tus nqi pib teeb tsa rau 1, BLOSUM45, tsis tshua muaj lim lim tau qhib, cov lus nug kev pab cuam lim tau teeb tsa rau 20 feem pua) siv cov lus nug LhKNOT. Cov cuab yeej Expasy (web.expasy.org/translate/; nkag rau 9 Lub Kaum Ob Hlis 2022) tau siv los txhais cov TSA sequences [75]. Cov txiaj ntsig tau kwv yees peptide sequences tau manually curated raws li muaj cov cysteine motif. Rau G. brasiliensis, AUGUSTUS-raws li (http://bioinf.uni-greifswald.de/augustus; nkag tau rau 9 Plaub Hlis 2020) [76] gene kwv yees los ntawm ib feem ntawm kev sib sau ua ke genomic (Gen-Bank: GCA{15} }.1) tau tsim los siv Nasonia vitripenis genome kev cob qhia module.
Cov txiaj ntsig tau kwv yees cov proteins tau tshawb fawb siv blastp (e-tus nqi pib tau teeb tsa rau 1, tsis tshua muaj lim lim tau qhib, cov lus nug kev pab cuam lim teeb rau 20 feem pua) tawm tsam LhKNOT. Alignments thiab visualization tau ua tiav siv EBI's Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo; nkag tau rau 9 Lub Kaum Ob Hlis 2022) [77] kom paub meej tias qhov muaj ntawm knottin motif thiab rau kev kho ntxiv ntawm cov kab ke.
Cov txheej txheem txheeb xyuas tau ua raws li LhKNOT los tsim kom muaj ntau qhov kev sib txuas ua ke siv T-COFFEE (https://www.ebi.ac.uk/Tools/msa/tcoffee; nkag mus rau 9 Kaum Ob Hlis 2022) [78] thiab pom kev siv ESPript3 (https://espript.ibcp.fr/ESPript/ESPript/index.php; nkag tau rau 9 Lub Kaum Ob Hlis 2022) [79] txhawm rau txheeb xyuas cov khoom seem thiab/lossis motif(s) hauv cov kab ke ua ke.
4.2. LhKNOT Primary Structure Analysis
Lub LhKNOT ib ntus kuj tau txheeb xyuas los ntawm domain/motif detection database Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd. shtml; accessed on 6 April 2018) thiab Pfam ( pfam.xfam.org; nkag mus rau lub Plaub Hlis 6, 2018) txhawm rau txheeb xyuas cov npe lossis cov npe sib txuas [80,81].
Cov kab ke tau txheeb xyuas ntxiv los ntawm kev siv cov teeb liab sib piv twv twv software SignalP{{0}}.0 (https://services.healthtech.dtu. dk/service.php?SignalP-5 .0; nkag mus rau 6 Plaub Hlis 2018) [82] thiab Phobius (https://www. ebi.ac.uk/Tools/pfa/phobias; nkag mus rau 6 Plaub Hlis 2018) [83] txhawm rau txheeb xyuas qhov chaw ntawm lub teeb liab sib lawv liag thiab tso cai rau kom meej meej tus yam ntxwv ntawm peptide. Cov peptide paub tab ntawm 36 amino acids (nrog rau kev kwv yees cov teeb liab sib lawv liag, residues 1-24, tshem tawm) tau siv rau kev tshuaj xyuas ntxiv.
Cov qauv thib ob ntawm peptide tau kwv yees siv cov meta-server Sympred (https://www.ibi.vu.nl/programs/sympredwww; nkag mus rau 6 Plaub Hlis 2018) [84] thiab txheeb xyuas kom paub meej tias nws cov lus teb rau qhov xav tau thib ob qauv rau knottin quav: thawj ntu alpha-helical ntu (xws li cov cim qhia ua ntu zus) ua raws li peb qhov kev sib piv ntawm beta strands.
4.3. Tertiary Structure Prediction and Evaluation of LhKNOT thiab Nws Homologs
Cov kev pab cuam I-Tasser (https://zhanggroup.org/I-TASSER; nkag rau 16 Plaub Hlis 2018) [85], Modeller (https://salilab.org/modeller; nkag rau 16 Plaub Hlis 2018) [{{7 }}], thiab HHPred (https://toolkit.tuebingen.mpg.de/tools/hhpred; nkag tau rau 6 Plaub Hlis 2018) [37] tau siv los ua qauv qauv tertiary ntawm LhKNOT. Peb cov qauv homologs nrog cov pob txha zoo li fold Alo-3 (PDB: 1Q3J) [30], omega-agatoxin-IVA (PDB: 1IVA) [28], thiab PAFP-S (PDB: 1DKC) [25] tau raug txheeb xyuas raws li cov neeg sib tw sab saum toj thiab siv los ua cov qauv hauv cov qauv tsim cov kev pab cuam.
Tus qauv kwv yees rau LhKNOT tau soj ntsuam los ntawm Verify3D (https://www.doe-mbi.ucla.edu/verify3d; nkag rau 18 Plaub Hlis 2018) [39], VoroMQA (https://bioinformatics.lt/wtsam/voromqa; nkag mus rau 18 Plaub Hlis 2018) [40], thiab Prosaweb (https://prosa.services.came.sbg.ac.at/prosa.php; nkag mus rau 18 Plaub Hlis 2018) [38]. Kev ntsuas peptides me me nrog cov cuab yeej ntsuas tus qauv tuaj yeem muaj kev txwv.
Yog li, cov qhab nia los ntawm kev daws teeb meem tertiary ntawm NMR thiab crystallography-los ntawm peptides ntawm qhov loj thiab zoo sib xws (PDB: 1Q3J, 1IVA, thiab 1DKC) tau siv los txhais cov qhab nia ntsuam xyuas ntawm peb cov qauv LhKNOT. Cov qauv saum toj kawg nkaus tau tsim los siv HHPred [37], ib qho kev qhia ua qauv uas siv qhov kev tsim qauv tsim qauv, Modeller [86] siv kev txwv-raws li kev sib piv qauv qauv. Tsis tas li ntawd, KNOTTIN database's KNOTER3D cuab tam (https://www.dsimb.inserm.fr/KNOTTIN/knoter3d.php; nkag mus rau 15 Tsib Hlis 2020) [36] tau siv los ntsuas tus qauv tus qauv tertiary, lees paub LhKNOT kev saws me nyuam. Knottin quav. Tag nrho cov homologs uas tsis paub cov qauv tau ua qauv siv cov qauv saum toj kawg nkaus uas tau txheeb xyuas hauv HHPred [37].
4.4. CPC Clip Motif Identification
Superposition ntawm LhKNOT tus qauv qauv nrog cov qauv paub ntawm CPC clip uas muaj knottin peptide los ntawm cactus Pereskia bleo (PDB: 5XBD) [26] siv cov kev pab cuam Superpose (http://superpose.wishartlab.com; nkag mus rau 12 Cuaj hlis 2{ {11}}18) [87] thiab MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt; nkag rau 12 Cuaj hlis 2018) [88] qhia txog qhov muaj cov heparin-binding CPC clip motif. Txhawm rau kom paub meej tias muaj CPC clip hauv LhKNOT, docking tsom xam siv qhov kev pab cuam, ClusPro 2.0 (uas muaj cov kev txwv tshwj xeeb rau heparin li ligand; https://cluspro.org; nkag mus rau 2 Lub Peb Hlis 2019) [89], thiab kev tshuaj xyuas tom qab ntawm kev kwv yees docking scenarios hauv qhov kev pom pom, PyMOL (https://pymol.org; nkag rau 10 Lub Peb Hlis 2019) [90] tau ua.
Tag nrho cov peev xwm docking scenarios tau raug tshuaj xyuas los txheeb xyuas cov khoom seem uas tsim cov CPC clip motif siv cov kauj ruam hauv qab no. Tag nrho cov polar kev sib cuam tshuam ntawm LhKNOT thiab heparin tau pom hauv PyMOL thiab cov npe ntawm cov koom nrog cov seem tau muab tso ua ke. Cov kev kwv yees uas muaj qhov tsim nyog Cation-Polar-Cation kev sib cuam tshuam tau raug tshuaj xyuas ntxiv rau kev ua raws li CPC clip motif parameter los ntawm kev ntsuas qhov deb ntawm alpha carbons ntawm kev koom nrog cov residues nrog rau qhov kev ncua deb ntawm qhov nruab nrab ntawm pawg ntawm cov khoom seem. PyMOL's "Measurements" Wizard tool tau siv los ntsuas qhov deb ntawm -carbons ntawm cov khoom seem uas koom nrog.
Txhawm rau ntsuas qhov kev ncua deb ntawm cov chaw nruab nrab ntawm pawg, tus neeg siv-tsim tsab ntawv muaj nyob rau ntawm Pymolwiki lub vev xaib (Henschel, https://pymolwiki.org/index.php/Center_ ntawm _mass; nkag mus rau ntawm 15 Lub Peb Hlis 2019) tau siv los tsim cov pseudo-atoms ntawm qhov ntsuas qhov nruab nrab ntawm pawg. PyMOL's APBS Electrostatics Plugin [91] tau siv los ua cov electrostatics saum npoo rau cov qauv LhKNOT. Cov kauj ruam zoo sib xws tau ua los txheeb xyuas CPC clip motif nyob rau hauv txhua qhov seem ntawm knottin proteins hauv Supplementary Table S5.
4.5. Association ntawm LhKNOT nrog Lwm Cov GAGs
Txhawm rau soj ntsuam seb CPC clip yuav pab txhawb kev khi nrog GAGs ntxiv, kev txuas ntxiv tau ua nrog hyaluronic acid (PDB: 1HYA) thiab keratan sulfate (PDB: 1KES). Nyob rau hauv cov ntaub ntawv ntawm hyaluronic acid, extraneous atoms (O, Na) raug tshem tawm ntawm qhov chaw nyob ib ncig ntawm lub molecule. Keratin sulfate tus qauv tau siv raws li yog. Docking tau ua tiav siv AutoDock (https://autodock.scripps.edu; nkag rau 7 Plaub Hlis 2020) [92], thiab cov txiaj ntsig tau pom hauv PyMOL. Cov txiaj ntsig tau muab tso rau hauv PDBSum (http://www.ebi.ac. uk/thornton-srv/databases/pdbsum/Generate.html; nkag mus rau 10 Plaub Hlis 2020) [93] los tsim Ligplots kom paub meej GAG-CPC clip binding .
4.6. Protein-Heparin Docking Analysis rau Mutated CPC Clip hauv LhKNOT
Txhawm rau tshawb xyuas qhov tseem ceeb ntawm CPC clip hauv heparin khi, cov qauv hloov pauv ntawm LhKNOT tau tsim los siv PyMOL's mutagenesis tool, hloov CPC clip seem nrog alanine. Cov qauv tsim tau tsim ib qho, thawj zaug hloov pauv tsuas yog R14, tom qab ntawd R1 thiab R14, thiab thaum kawg R14, R1, thiab S3. Heparin docking ntawm txhua tus qauv tau ua tiav siv ClusPro, thiab kev tshuaj xyuas tau ua raws li tau piav qhia dhau los.
4.7. Protein-Heparin Docking Analysis hauv Cov Qauv Homologs thiab Drosomycin
Txhawm rau soj ntsuam ntxiv qhov muaj CPC clip motif hauv tsev neeg knottin peptide, cov txheej txheem dhau los tau nthuav dav ntxiv rau (a) homologs ntawm LhKNOT (PDB: 5XBD, 1Q3J, 1IVA, thiab 1DKC) [25,26,28,30 ]; (b) Drosophila Drosomycin (PDB: 1MYN) [43], peptide uas paub txog kev tiv thaiv kab mob; (c) qauv homologous knottin proteins thiab (d) tus neeg sawv cev knottins los ntawm ntau pawg hauv KNOTTIN database. Cov txiaj ntsig tau raug txiav txim siab ua raws li cov qauv yog tias kev ntsuas poob hauv 3Å ntawm CPC clip tsis, kom tso cai rau qee qhov kev hloov pauv ntawm cov voj voog protein.
Cov khoom siv ntxiv:
Cov ntaub ntawv txhawb nqa hauv qab no tuaj yeem rub tawm ntawm https://www.mdpi.com/article/10.3390/pathogens12010143/s1. Tab S1. Txheeb xyuas homologs ntawm LhKNOT siv cov txheej txheem sib txawv. Tab S2. Putative L. heterotoma homologs ntawm LhKNOT. Cov kab ke raug txheeb xyuas los ntawm tblastn los ntawm peb Lh14 cov ntaub ntawv sau tseg (saib Cov Txheej Txheem rau cov lej nkag).
Cov ntawv sau ua ke los ntawm cov hlwb uas muaj duab ntxoov ntxoo encode zoo tib yam knottin peptides. Cov lej hloov pauv tau pom nyob rau hauv bold encode ob lub peptides sib txawv. Cov txiaj ntsig tau muab rov qab los ntawm Cov Lus piav qhia muaj los ntawm NCBI cov txiaj ntsig tawg. Tab S3. Putative L. boulardi knottin homologs of LhKNOT. Cov txheej txheem raug txheeb xyuas los ntawm tblastn los ntawm plaub L. boulardi transcriptome datasets (saib Cov Txheej Txheem rau cov lej nkag). Zoo tib yam knottin peptides muab tau los ntawm cov ntawv sau sib txawv yog pawg los ntawm cov xim. Cov txiaj ntsig tau muab los ntawm Cov Lus Qhia Cov Lus Qhia los ntawm NCBI cov txiaj ntsig tawg. Tab S4.
Putative knottin sequences los ntawm Ganaspis spp., homologous rau LhKNOT. G. hookeri sequences tau txheeb xyuas nrog tblastn, thaum G. brasiliensis sequences tau txheeb xyuas nrog lub moj tej tawg. Cov txiaj ntsig rau G. hookeri thiab G. brasiliensis tau muab los ntawm Cov Lus Qhia piav qhia muaj los ntawm NCBI tshawb nrhiav. Shaded sequences encode zoo tib yam peptides. Tab S5.
Ib daim ntawv teev cov amino acid residues hauv CPC clips ntawm kev tshuaj xyuas paub paub knottin, qauv knottin peptides, thiab Drosomycin. Kev ntsuas qhov deb (Å) ntawm alpha-carbon thiab sidechain center ntawm pawg (ntawm pseudo-atom) ntawm kev koom tes residues ntawm CPC clip motifs tau qhia. Qhov tseem ceeb: ntxiv rau paub kev ua phem knottins (toxins), Φ hauv silico qauv, * non-knottin quav, ± lwm yam putative peptide, highlighted ntsuab thiab daj rau demarcate peptides tshwm sim los ntawm tib daim ntawv sau tseg. Daim duab S1: (A) Prosa-web evaluation profile. Z-cov qhab nia ntawm tus qauv yog sawv cev raws li cov ntsiab lus dub ntawm lub nraub qaum ntawm Z-cov qhab nia ntawm cov qauv paub los ntawm PDB (sab laug) thiab kev pom ntawm peb-dimensional qauv LhKNOT nrog cov seem xim ntawm xiav mus rau liab nyob rau hauv qhov kev txiav txim ntawm nce. residue zog (txoj cai). (B) Txheeb xyuas qhov profile ntsuas. Cov phiaj xwm qhia qhov nruab nrab thiab nyoos 1D-3D cov qhab nia rau LhKNOT thiab lub vaj huam sib luag saum toj sau cov feem pua ntawm cov seem uas tau qhab nia nruab nrab ntawm 0.2 lossis siab dua.
Tus qauv dhau los muaj tsawg kawg yog 80 feem pua ntawm nws cov seem tau qhab nia siab dua 0.2. (C) VoroMQA evaluation profile. VoroMQA cov qhab nia thoob ntiaj teb hauv cov ntsiab lus ntawm cov qhab nia zoo X-ray qauv (sab laug) thiab cov phiaj xwm ntawm cov qhab nia hauv zos thiab tag nrho cov qhab nia rau LhKNOT (txoj cai). Daim duab S2. Ligplot uas qhia txog kev sib cuam tshuam ntawm LhKNOT nrog keratin sulfate (PDB: 1KES) suav nrog CPC clip residues (R1-S3-R14). Green dashed kab qhia txog kev sib cuam tshuam ntawm hydrogen. Daim duab S3. Ligplot qhia kev sib cuam tshuam ntawm LhKNOT nrog hyaluronic acid (PDB: 1HYA) suav nrog CPC clip residues (R1-S3-R14).
Green dashed kab qhia txog kev sib cuam tshuam ntawm hydrogen. Daim duab S4. (A) Ligplot qhia kev sib cuam tshuam ntawm cov tsiaj qus LhKNOT cuam tshuam nrog heparin ntawm thawj CPC clip (R1-S3-R14). (B) Ligplot uas qhia txog kev sib cuam tshuam ntawm mutant R14A LhKNOT cuam tshuam nrog heparin ntawm CPC txuas ntxiv (R1-S3-R30). Green dashed kab qhia txog kev sib cuam tshuam ntawm hydrogen. Cov ntaub ntawv pov thawj [94–111] raug muab tso rau hauv Cov Khoom Siv Ntxiv.
Tus sau kev koom tes:
Conceptualization, SS, SG, thiab JA; methodology, JA, thiab SS; validation, SS, SG, JA, JL, thiab LVC; kev soj ntsuam, JA, JL thiab LVC; kev tshawb nrhiav, JA, JL, thiab LVC; cov peev txheej, SS thiab SG; data curation, JA, JL, thiab LVC; kev sau ntawv-kev npaj ua ntej, JA; sau ntawv- tshuaj xyuas thiab kho, SS, SG, JA, JL thiab LVC; visualization, JA, thiab JL; kev saib xyuas, SS thiab SG; kev tswj xyuas qhov project, SS thiab SG Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Nyiaj txiag:
Qhov kev tshawb fawb no tsis tau txais nyiaj txiag.
Institutional Review Board Statement:
Tsis siv tau.
Cov Lus Qhia Txog Kev Pom Zoo:
Tsis siv tau.
Cov ntaub ntawv muaj nyob:
Cov ntaub ntawv qhia hauv qhov kev tshawb fawb no muaj nyob rau hauv kab lus thiab Cov Khoom Siv Ntxiv.
Kev lees paub:
Peb ua tsaug rau Brian Wey rau lawv txoj kev pab nrog G. brasiliensis noob kwv yees thiab SA Tasnim Ahmed rau nws kev pab nrog formatting cov duab.
Kev tsis sib haum xeeb ntawm kev txaus siab:
Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.
Cov ntaub ntawv
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