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Biochemistry

X-Ray krystallografi at studere oligomeric stat Overgang af Thermotoga maritima M42 Aminopeptidase TmPep1050

Published: May 13, 2020
doi:
10.3791/61288
, , ,
1Laboratory of Microbiology, Department of Molecular Biology,Université Libre de Bruxelles, 2Labiris Institut de Recherche

Summary

Denne protokol er udviklet til at studere dimer-dodecamer overgang af TmPep1050, en M42 aminopeptidase, på det strukturelle niveau. Det er en enkel rørledning, der starter fra proteinrensning til røntgendatabehandling. Crystallogenesis, datasæt indeksering, og molekylær udskiftning er blevet understreget gennem et studie, TmPep1050H60A H307A variant.

Abstract

Den M42 aminopeptidases form funktionelt aktive komplekser lavet af 12 underenheder. Deres samling proces synes at være reguleret af deres metal ion cofaktorer udløser en dimer-dodecamer overgang. Ved metalionbinding sker der flere strukturelle ændringer på det aktive sted og ved interaktionsgrænsefladen, der former dæmninger for at fremme selvsamlingen. For at observere sådanne ændringer skal stabile oligomerer isoleres forud for strukturundersøgelsen. Rapporteret her er en metode, der tillader rensning af stabile dodecamers og dimers af TmPep1050, en M42 aminopeptidase af T. maritima, og deres struktur bestemmelse ved X-ray krystallografi. Dimers blev fremstillet af dodecamers ved at fjerne metalioner med et chelaterende middel. Uden deres cofactor blev dodecamers mindre stabile og blev fuldt adskilt ved opvarmning. De oligomeriske strukturer blev løst ved hjælp af den enkle molekylære udskiftningsmetode. For at illustrere metoden præsenteres strukturen af en TmPep1050-variant, der er fuldstændig svækket i metalionbinding, og som ikke viser nogen yderligere opdeling af dimers til monomerer.

Introduction

Oligomerization er en fremherskende proces, der dikterer de biologiske funktioner af mange proteiner. I Escherichia colianslås det, at kun 35 % af proteinerne er monomeriske1. Nogle proteiner, kaldet morfeeeiner, kan endda vedtage flere oligomeriske tilstande med underenheder med særskilt struktur i hver oligomeric tilstand2. Overgangen mellem deres oligomeriske tilstande er ofte et middel til at regulere protein aktivitet som hver oligomeric tilstand kan have en anden specifik aktivitet eller funktion. Flere eksempler på morfeseiner er veldokumenteret i litteraturen, navnlig porphobilinogensyntase3, HPr kinase/phosphatase4, Lon protease5, laktat dehydrogenase6, glyceraldehyd-3-phosphat dehydrogenase7, pyruvatkinase8, citratsyntetase9og ribonuclease A10. For nylig har vi beskrevet M42 aminopeptidase TmPep1050, et andet eksempel på enzym med morfemein-lignende adfærd, hvis aktivitet afhænger af dens oligomeric stater11. Overgangen mellem dens oligomeriske tilstande er medieret af dens metalliske cofaktorer, som fremkalder flere strukturelle ændringer af underenheder.

Den M42 aminopeptidase familien tilhører MH klan12,13, og er udbredt blandt bakterier og Archaea14. M42 aminopeptidases er ægte dinuclear enzymer nedbrydende peptider op til 35 aminosyre rester i længde15. De vedtager en ejendommelig tetrahedron-formet struktur lavet af 12 underenheder med deres aktive steder orienteret mod en indre hulrum. Et sådant arrangement beskrives ofte som en nanoopdelte aktivitet for at undgå ukontrolleret proteolyse. Den fysiologiske funktion af M42 aminopeptidases kan være forbundet med proteasom, hydrolyserende peptider som følge af proteinnedbrydning 16,17. Pyrococcus horikoshii har fire M42 aminopeptidases, hver præsenterer forskellige, men komplementærespecificiteter 18,19,20,21. Ental, heterocomplexes lavet af to forskellige typer af underenheder er blevet beskrevet i P. horikoshii, hvilket tyder på eksistensen af peptidasome komplekser22,23.

Flere strukturer af M42 aminopeptidaser er blevet beskrevet ilitteraturen 11,16,18,19,20,24,25,26. Underenheden består af to forskellige domæner, et katalytisk domæne og et dimeriseringsdomæne. Den katalytiske domæne vedtager en fælles α/ β fold bevaret i hele MH klan, den arketypiske katalytiske domæne er aminopeptidase Ap1 af Vibrio proteolyticus27. Den dimerization domæne vedtager en PDZ-lignende fold16 og kan have, ud over sin rolle i oligomerization, en rolle i at kontrollere substrat adgang og bindende i det indre hulrum11. Da den grundlæggende byggesten er en dimer, dodecamer er ofte beskrevet som sammenslutningen af seks dimers, hver dimer er placeret på hver kant af tetrahedron16. Den oligomerization af M42 aminopeptidases bygger på tilgængeligheden af sine metal cofaktorer. Divalente metalioner, ofte Zn2+ og Co2+,er katalytisk involveret i peptidbindingen og hydrolyse. De findes i to forskellige bindingssteder, nemlig M1 og M2 sites. De to metalioner også køre og finjustere oligomerization som påvist for PhTET2, PhTET3, PfTET3, og TmPep105011,24,28,29. Når metal cofaktorer er opbrugt, dodecamer demonterer i dimers, ligesom i PhTET2, PhTET3, og TmPep105011,16,28, eller endda monomerer, som i PhTET2 og PfTET324,29.

Præsenteret her er en protokol, der anvendes til at studere strukturerne i TmPep1050 oligomers. Denne protokol er et sæt af almindelige metoder, herunder proteinrensning, proteolytisk aktivitet screening, krystallisering, X-ray diffraktion, og molekylær udskiftning. Finesser iboende beskæftiger sig med metalloenzymer, protein oligomerisering, protein krystallisering og molekylær udskiftning understreges. Et studieeksemier præsenteres også for at vise, om TmPep1050 dodecamers yderligere kan dissociate i monomerer eller ej. For at løse dette spørgsmål, en TmPep1050 variant, TmPep1050H60A H307A, er blevet undersøgt, hvis metal bindingssteder er svækket ved at mutere His-60 (M2 site) og His-307 (M1 site) til Ala rester. Denne protokol kan indkvarteres til at studere andre M42 aminopeptidases eller metalloenzymer med morfem-lignende adfærd.

Protocol

1. Produktion og rensning af rekombinant TmPep1050 BEMÆRK: Herefter beskrives kloningsproceduren og rensningen af vildtypen TmPep1050 tilpasset fra en tidligere undersøgelse11. Alternativt kan kloning gøres ved hjælp af et syntetisk gen. For at generere TmPep1050 varianter, site rettet mutagenesis kan udføres efter, for eksempel, single-primer reaktioner i parallel protokol (SPRINP) metode30. Renselsesprotokollen kan bruges til TmPep1050 varia…

Representative Results

For at studere en mulig dodecamer dissociation i monomerer i TmPep1050, hans-60 og Hans-307 codons blev erstattet af alanin codon ved hjælp af et syntetisk gen. Dette gen blev derefter klonet i pBAD vektor for udtryk og rensning af den tilsvarende TmPep1050 variant efterfølgende opkaldt TmPep1050H60A H307A. Størrelseseksklusivekromatografi (Figur 3B) viste, at det rensede protein havde en tilsyneladende molekylvægt på 56 kDa (molekylvægt af monomeren er 36,0 kDa). En lignend…

Discussion

Den protokol, der er beskrevet heri, gør det muligt at forstå overgangen til dimer-dodecamer af TmPep1050 på det strukturelle niveau. Metoden blev tidligere oplevet til bestemmelse af strukturen af både TmPep1050 oligomers11. Det mest udfordrende skridt var at finde betingelser, der fremmer dissociation af dodecamers i stabile dimers. Sådanne forhold måtte være milde nok til at gøre det muligt at reassociere dimers til dodecamers, når metal ion cofactor blev tilføjet. Adskillelsen af oli…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Martine Roovers for korrekturlæsning af dette papir og med konstruktive kommentarer. Adgang til Proxima 2 beamline (SOLEIL synchrotron) var inden for Blokallokeringsgrupper 20151139.

Materials

1,10-phenanthroline Sigma-Aldrich 13, 137-7
Amicon Ultra 0.5 ml Centrifugal Filters Ultracel 30K Merck Millipore UFC503096
Amicon Ultra 15 Centrifugal Filters Ultracel 30K Merck Millipore UFC903024
Benzonase Nuclease Merck Millipore 70664-3
CCP4 N/A visit http://www.ccp4.ac.uk/
cOmplete EDTA-free Roche 5056489001
Coot N/A visit https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Crystal Screen I Hampton Research HR2-110
Crystal Screen II Hampton Research HR2-112
DreamTaq Green PCR Master Mix ThermoFisher Scientific K1082
EasyXtal 15-well tool NeXtal 132007
Escherichia coli PPY strain N/A see reference 31
Escherichia coli XL1 blue strain Agilent 200249
Gel Filtration Calibration Kit HMW GE Healthcare Life Sciences 28-4038-42
Gel Filtration Calibration Kit LMW GE Healthcare Life Sciences 28-4038-41
Gel Filtration Standard Biorad 1511901
GeneJET Plasmid Miniprep Kit ThermoFisher Scientific K0503
Index Hampton Research HR2-144
Litholoops Molecular Dimensions
L-leucine-p-nitroanilide Bachem AG 40010720025
Natrix 1 Hampton Research HR2-116
Natrix 2 Hampton Research HR2-117
Neggia plugin Dectris N/A visit https://www.dectris.com/
NeXtal Tubes JCSG Core Suite I NeXtal 130724
NeXtal Tubes JCSG Core Suite II NeXtal 130725
NeXtal Tubes JCSG Core Suite III NeXtal 130726
NeXtal Tubes JCSG Core Suite IV NeXtal 130727
pBAD-TOPO ThermoFisher Scientific K430001
Phenix N/A visit https://www.phenix-online.org/
Phusion High-Fidelity DNA polymerase ThermoFisher Scientific F-530L
Salt RX 1 Hampton Research HR2-107
Salt RX 2 Hampton Research HR2-109
SnakeSkin Dialysis Tubing, 3.5K MWCO ThermoFisher Scientific 88242
Source 15Phe GE Healthcare Life Sciences 17014702
Source 15Q GE Healthcare Life Sciences 17094705
Superdex 200 prep grade GE Healthcare Life Sciences 17104301
Thermotoga maritima MSB8 strain American Type Culture Collection ATCC 43589
TmCD00089984 DNASU Plasmid Repository N/A
XDS N/A visit http://xds.mpimf-heidelberg.mpg.de/
xdsme N/A visit https://github.com/legrandp/xdsme
DOWNLOAD MATERIALS LIST

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Tags

Keywords: X-ray CrystallographyOligomeric StateThermotoga MaritimaAminopeptidaseTmPep1050Activity AssayApoenzyme PreparationDialysisUltrafiltration
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Dutoit, R., Brandt, N., Van Elder, D., Droogmans, L. X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050. J. Vis. Exp. (159), e61288, doi:10.3791/61288 (2020).
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