Bacterial Multidrug Exporters: Methods and Protocols

351 Pages·2018·10.171 MB·English

by  Akihito Yamaguchi,Kunihiko Nishino (eds.)

Methods in
Molecular Biology 1700
Akihito Yamaguchi
Kunihiko Nishino Editors
Bacterial
Multidrug
Exporters
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
Bacterial Multidrug Exporters
Methods and Protocols
Edited by
Akihito Yamaguchi
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan
Kunihiko Nishino
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan;
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
Editors
AkihitoYamaguchi KunihikoNishino
InstituteofScientificandIndustrial InstituteofScientificandIndustrial
Research,OsakaUniversity Research,OsakaUniversity
Ibaraki,Osaka,Japan Ibaraki,Osaka,Japan
GraduateSchool
ofPharmaceuticalSciences,OsakaUniversity
Suita,Osaka,Japan
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-7452-8 ISBN978-1-4939-7454-2 (eBook)
https://doi.org/10.1007/978-1-4939-7454-2
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Preface
Multidrug exporters are intrinsic membrane proteins widely distributed in bacteria. They
play as cellular self-defense mechanisms and have some other physiological roles. They
contribute to bacterial tolerance against antibiotics. When they are over-expressed, they
cause multidrug resistance. Multidrug-resistant pathogens have caused great difficulties in
modern chemotherapy. There have been no clinically useful drugs against bacterial multi-
drug exporters. They can be classified into three major families: ABC, MFS, and RND.
ABC-type exporters are ATP-hydrolysis-coupled transporters, which are major multidrug
exporters in mammalian models,but in microorganisms, ABC-type exporters play a minor
role in drug resistance. MFS-type exporters are drug/proton antiporters, which are major
drug exporters in multidrug resistant Gram-positive pathogens. MATE-type is a subfamily
of MFS-type transporters. RND-type exporters are multidrug exporters characteristic in
Gram-negative bacteria, which consist of a tripartite complex passing through cytoplasmic
membrane, periplasm, and the outer membrane. The most characteristic common proper-
ties of multidrug exporters are their extremely broad substrate specificity. For example,
major multidrug exporter AcrAB-TolC in E. coli exports both aromatic and aliphatic
compounds including cationic, anionic, twitter ionic, and neutral compounds. However,
it has own specificity. It exports oxacillin but does not export carbenicillin. Clarifying the
mechanismofmultidrugexporters,specificallyastheyexportsuchawiderangeofdrugsand
toxiccompounds,isoneofmajorchallengesposedtoour modernscience.
Elucidation of multidrug efflux mechanisms has been greatly advanced mainly by
structure determination of bacterial multidrug exporters during the last decade. Prior to
the2000s,notransporters’molecularstructuresweresolved.Now,thecrystalstructuresof
more than ten multidrug exporters including all three types (RND, MFS, and ABC) have
been solved. This success is supported by the advancement of the technology to express a
largeamountoftaggedmembraneproteins,developmentofdetergentstosolubilizemem-
brane proteins, large-scale purification using affinity chromatography, as well as develop-
mentofthemethodforcrystallizationincludingfacilitatorofcrystallizationsuchasDARPin
and monobody. As a result, we have been able to understand the outline of the structural
basisofmultidrugrecognitionandmultidrugeffluxmechanisms.
In the first part of this book, we present protocols to introduce marvelous success in
determining multidrug exporter structures during the last decade. Chapters 1 and 2 show
the protocols for determination of the high-resolution structures and the ligand-binding
structures of RND-type multidrug exporter AcrB, which are the pioneering works for the
structuralstudyofbacterialmultidrugexporters.Theyrevealedthatthemultidrugeffluxis
mediated by the functional rotation mechanism of an asymmetric homo-trimer, and the
structural basis of the multidrug recognition is a multisite drug binding. RND-type trans-
portersaretripartitecomplexescomposedofcellmembranetransporters,outermembrane
channels, and adaptor proteins. Chapter 3 describes the crystal structure of another inter-
estingtypemultidrugexporter,MATE-type,boundwithitsinhibitors.Italsodescribesthe
novelartificialand systematictransporter-inhibitorconstruction method.Chapter4shows
the first crystal structure of the complex of a cell membrane transporter and an adaptor
proteintwo-partcomplex.Unlikelytotheconventionalsense,anadaptorproteinisnotjust
an adaptor but the hexamer of the adaptor proteins makes a periplasmic channel that
v
vi Preface
connectsbetweenacellmembranetransporterandanoutermembranechannel.Chapter5
shows the first whole structure of a tripartite complex of the RND-type exporter by cryo-
EM images. Chapter 6 shows another strong tool for structure determination of a mem-
branetransporter usingNMR spectroscopy.Chapter 7describesone ofthe powerfultools
forcrystallizationofamembranetransporter.
Inordertounderstandhowtoworkthemolecularstructuresofmultidrugexportersto
transport drugs, biochemical and bioengineering analysis is absolutely necessary. In the
second section, we will show a few important examples of the numerous biochemical and
genetic studies of exporters. With respect to the RND-type transporters, reconstitution
studies for whole tripartite complex are tremendously difficult because the complex pene-
trates through two membranes. Chapter 8 shows the breakthrough achievement of the
reconstitution of the tripartite complex and the measurement of multidrug transport by
RND-type transporters through artificial membranes. Chapter 9 proves the functional
rotation mechanism of drug export mediated by RND-type exporters using artificial cova-
lentlylinkedtrimersofcellmembranetransporter.Chapter10showstheintra-proteindrug
translocationpathwaybyintroducingsite-directedmutagenesis.
In PartIII, weshow thecomputationalanalysisofhow toworktheexporterstructure
and how to predict a novel efflux pump. Chapter 11 shows an excellent example of the
molecular dynamic simulations of the RND-type exporter. Chapter 12 shows a transcrip-
tomicapproachtoidentifynoveleffluxpumps.
Multidrug exporters play a variety of physiological roles under various expression
controls. In Part IV, we show how to regulate the exporter expression and biomedical
rolesofexporters.Chapters13and14showtheconditionandthemechanismofexpression
regulation ofthe bacterial multidrug exporters. Chapter 15shows the identification ofthe
expression regulators. Chapter 16 shows high-throughput screening of multidrug efflux
systemusefulforidentificationofthephysiologicalroleofexporters.
Finally, in the Part V, we show the advanced technologies useful for future works of
multidrug exporters. Chapter 17 shows single-molecule analysis of membrane transporter
activityusingartificialmembranesstretchedonamicrochamber.Chapter18showsasingle-
cell efflux assay method using femtoliter droplet arrays. Chapter 19 shows reconstitution
and active transport assay using liposomes including V-ATPase for a proton-motive force
supplier.
The field of multidrug exporter studies is a fast developing field of science. I hope this
collectionofprotocolswillcontributetotheworkofmanyresearchersstudyingmultidrug
exporters.Iwouldliketothankallauthorsforsharingtheirvaluableexperienceandinsights
withtheresearchcommunityatlarge.IwouldliketothanktheserieseditorDr.JohnWalker
forhelpwithreviewingthebook.
Ibaraki,Osaka,Japan AkihitoYamaguchi
KunihikoNishino
Contents
Preface ..................................................................... v
Contributors................................................................. ix
PART I STRUCTURAL ANALYSIS OF BACTERIAL MULTIDRUG EXPORTERS
1 High-ResolutionCrystallographicAnalysisofAcrBUsingDesigned
AnkyrinRepeatProteins(DARPins)....................................... 3
HengKeatTam,VivekaNandMalviya,andKlaasM.Pos
2 CrystallographicAnalysisofDrugandInhibitor-BindingStructure
ofRND-TypeMultidrugExporterAcrBinPhysiologicallyRelevant
AsymmetricCrystals..................................................... 25
RyosukeNakashima,KeisukeSakurai,andAkihitoYamaguchi
3 CrystallographicAnalysisofMATE-TypeMultidrugExporter
withItsInhibitors....................................................... 37
TsukasaKusakizako,YoshikiTanaka,ChristopherJ.Hipolito,
HiroakiSuga,andOsamuNureki
4 CrystallographicAnalysisoftheCusBAHeavy-MetalEffluxComplex
ofEscherichiacoli ....................................................... 59
JaredA.DelmarandEdwardW.Yu
5 PurificationofAcrAB-TolCMultidrugEffluxPump
forCryo-EMAnalysis ................................................... 71
DijunDu,ZhaoWang,WahChiu,andBenF.Luisi
6 NMRSpectroscopyApproachtoStudytheStructure,Orientation,
andMechanismoftheMultidrugExporterEmrE........................... 83
MaureenLeningerandNathanielJ.Traaseth
7 GenerationofConformation-SpecificAntibodyFragments
forCrystallizationoftheMultidrugResistanceTransporterMdfA ............ 97
FrankJaenecke,YoshikoNakada-Nakura,KumarNagarathinam,
SatoshiOgasawara,KehongLiu,YunhonHotta,SoIwata,
NorimichiNomura,andMikioTanabe
PART II BIOCHEMICAL AND BIOENGINEERING ANALYSIS
OF BACTERIAL MULTIDRUG EXPORTERS
8 BiochemicalReconstitutionandCharacterization
ofMulticomponentDrugEffluxTransporters.............................. 113
MartinPicard,ElenaB.Tikhonova,IsabelleBroutin,ShuoLu,
AliceVerche`re,andHelenI.Zgurskaya
9 CovalentlyLinkedTrimersofRND(Resistance-Nodulation-Division)
EffluxTransporterstoStudyTheirMechanismofAction:
EscherichiacoliAcrBMultidrugExporterasanExample..................... 147
HiroshiNikaido
vii
viii Contents
10 DeterminingLigandPathThroughaMajorDrugTransporter,
AcrB,inEscherichiacoli.................................................. 167
FasahathHusainandHiroshiNikaido
PART III COMPUTATIONAL ANALYSIS OF BACTERIAL MULTIDRUG EXPORTERS
11 MolecularModelingofMultidrugPropertiesofResistance
NodulationDivision(RND)Transporters ................................. 179
PierpaoloCacciotto,VenkataK.Ramaswamy,GiulianoMalloci,
PaoloRuggerone,andAttilioV.Vargiu
12 ATranscriptomicApproachtoIdentifyNovelDrugEfflux
PumpsinBacteria....................................................... 221
LipingLi,SashaG.Tetu,IanT.Paulsen,andKarlA.Hassan
PART IV BIOMEDICAL APPROACH FOR BACTERIAL MULTIDRUG EXPORTERS
13 RegulationoftheExpressionofBacterialMultidrugExporters
byTwo-ComponentSignalTransductionSystems .......................... 239
KunihikoNishino
14 StudyoftheExpressionofBacterialMultidrugEffluxPumps
inAnaerobicConditions................................................. 253
JingjingSun,ZiqingDeng,DannyKaChunFung,andAixinYan
15 IdentificationofaStaphylococcusaureusEffluxPumpRegulator
UsingaDNA–ProteinAffinityTechnique.................................. 269
QueChiTruong-BolducandDavidC.Hooper
16 High-ThroughputFlowCytometryScreeningofMultidrug
EffluxSystems.......................................................... 293
MarkK.Haynes,MatthewGarcia,RyanPeters,AnnaWaller,
PietroTedesco,OlegUrsu,CristianG.Bologa,RadleighG.Santos,
ClemenciaPinilla,TerryH.Wu,JulieA.Lovchik,TudorI.Oprea,
LarryA.Sklar,andGeorgeP.Tegos
PART V ADVANCED TECHNOLOGIES EXPECTED FOR APPLICATION
TO MULTIDRUG EFFLUX TRANSPORT STUDIES
17 Single-MoleculeAnalysisofMembraneTransporterActivity
byMeansofaMicrosystem .............................................. 321
RikiyaWatanabe,NaokiSoga,Shin-yaOhdate,andHiroyukiNoji
18 Large-ScaleFemtoliterDropletArrayforSingleCellEffluxAssay
ofBacteria ............................................................. 331
RyotaIino,ShouichiSakakihara,YoshimiMatsumoto,
andKunihikoNishino
19 ReconstitutionandTransportAnalysisofEukaryoticTransporters
inthePost-GenomicEra ................................................ 343
HiroshiOmoteandYoshinoriMoriyama
Index ...................................................................... 353
Contributors
CRISTIANG.BOLOGA (cid:1) DivisionofTranslationalInformatics,DepartmentofInternal
Medicine,UniversityofNewMexicoSchoolofMedicine,Albuquerque,NM,USA
ISABELLEBROUTIN (cid:1) LaboratoiredeCristallographieetRMNBiologiques,UMR8015,
CNRS,Universite´ParisDescartes,Faculte´dePharmaciedeParis,Paris,France
PIERPAOLOCACCIOTTO (cid:1) DepartmentofPhysics,UniversityofCagliari,Monserrato,
CA,Italy
WAHCHIU (cid:1) NationalCenter forMacromolecularImaging,VernaandMarrsMcLean
DepartmentofBiochemistryandMolecularBiology,BaylorCollegeofMedicine,Houston,
TX,USA
JAREDA.DELMAR (cid:1) DepartmentofPhysicsandAstronomy,IowaStateUniversity,Ames,
IA,USA
ZIQING DENG (cid:1) SchoolofBiologicalSciences,TheUniversityofHongKong,Pokfulam,
HongKongSAR
DIJUNDU (cid:1) DepartmentofBiochemistry,UniversityofCambridge,Cambridge,UK
DANNYKACHUN FUNG (cid:1) SchoolofBiologicalSciences,TheUniversityofHongKong,
Pokfulam,HongKongSAR;DepartmentofBacteriology,Universityof
Wisconsin-Madison,Madison,WI,USA
MATTHEWGARCIA (cid:1) Center forMolecularDiscovery,UniversityofNewMexicoSchool
ofMedicine,Albuquerque,NM,USA;DepartmentofPathology,UniversityofNewMexico
SchoolofMedicine,Albuquerque,NM,USA
KARLA.HASSAN (cid:1) DepartmentofChemistryandBiomolecularSciences,Macquarie
University,NorthRyde,NSW,Australia
MARKK.HAYNES (cid:1) Center forMolecularDiscovery,UniversityofNewMexicoSchool
ofMedicine,Albuquerque,NM,USA;DepartmentofPathology,Universityof
NewMexicoSchoolofMedicine,Albuquerque,NM,USA
CHRISTOPHERJ.HIPOLITO (cid:1) DepartmentofChemistry,GraduateSchoolofScience,
TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan;FacultyofMedicine,University
ofTsukuba,Tsukuba,Japan
DAVIDC.HOOPER (cid:1) DivisionofInfectiousDiseasesandMedicalServices,Massachusetts
GeneralHospital,HarvardMedicalSchool,Boston,MA,USA
YUNHONHOTTA (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,Kyoto
University,Kyoto,Japan
FASAHATHHUSAIN (cid:1) DoctorEvidence,LLC,SantaMonica,CA,USA;Department
ofMolecularandCellBiology,UniversityofCalifornia,Berkeley,CA,USA;
VeteransAffairs,LosAngeles,CA,USA
RYOTAIINO (cid:1) OkazakiInstituteforIntegrativeBioscience,InstituteforMolecularScience,
NationalInstitutesofNaturalSciences,Okazaki,Aichi,Japan;DepartmentofFunctional
MolecularScience,SchoolofPhysicalSciences,TheGraduateUniversityforAdvanced
Studies(SOKENDAI),Kanagawa,Japan
SOIWATA (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,
Kyoto,Kyoto,Japan;ResearchAccelerationProgram,MembraneProteinCrystallography
Project,JapanScienceandTechnologyAgency,Sakyo-ku,Kyoto,Japan;ERATO,Iwata
ix
x Contributors
HumanReceptorCrystallographyProject,JapanScienceandTechnologyAgency,Sakyo-ku,
Kyoto,Japan;RIKEN,SPring-8Center,Sayo,Hyogo,Japan
FRANKJAENECKE (cid:1) HALOmem,MembraneProteinBiochemistry,Martin-Luther-University
Halle-Wittenberg,Halle(Saale),Germany
TSUKASAKUSAKIZAKO (cid:1) DepartmentofBiologicalSciences,GraduateSchoolofScience,
TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan
MAUREENLENINGER (cid:1) DepartmentofChemistry,NewYorkUniversity,NewYork,NY,USA
LIPINGLI (cid:1) DepartmentofChemistryandBiomolecularSciences,MacquarieUniversity,
Sydney,NSW,Australia
KEHONG LIU (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,
Kyoto,Kyoto,Japan
JULIEA.LOVCHIK (cid:1) Center forInfectiousDiseaseandImmunity,DepartmentofInternal
Medicine,UniversityofNewMexicoSchoolofMedicine,Albuquerque,NM,USA
SHUOLU (cid:1) DepartmentofChemistryandBiochemistry,UniversityofOklahoma,Norman,
OK,USA
BENF.LUISI (cid:1) DepartmentofBiochemistry,UniversityofCambridge,Cambridge,UK
GIULIANOMALLOCI (cid:1) DepartmentofPhysics,UniversityofCagliari,Monserrato,CA,Italy
VIVEKANANDMALVIYA (cid:1) InstituteofBiochemistry,GoetheUniversityFrankfurt,Frankfurt,
Germany
YOSHIMI MATSUMOTO (cid:1) DepartmentofBiomolecularScienceandRegulation,Institute
ofScientificandIndustrialResearch,OsakaUniversity,Osaka,Japan
YOSHINORIMORIYAMA (cid:1) DepartmentofMembraneBiochemistry,OkayamaUniversity
GraduateSchoolofMedicine,DentistryandPharmaceuticalSciences,Okayama
University,Okayama,Japan
KUMARNAGARATHINAM (cid:1) HALOmem,MembraneProteinBiochemistry,Martin-Luther-
UniversityHalle-Wittenberg,Halle(Saale),Germany
YOSHIKONAKADA-NAKURA (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,Kyoto
University,Kyoto,Kyoto,Japan;ResearchAccelerationProgram,MembraneProtein
CrystallographyProject,JapanScienceandTechnologyAgency,Sakyo-ku,Kyoto,Japan
RYOSUKENAKASHIMA (cid:1) DepartmentofCellMembraneBiology,InstituteofScientific
andIndustrialResearch,OsakaUniversity,Ibaraki,Osaka,Japan
HIROSHINIKAIDO (cid:1) DepartmentofMolecularandCellBiology,UniversityofCalifornia,
Berkeley,Berkeley,CA,USA
KUNIHIKONISHINO (cid:1) InstituteofScientificandIndustrialResearch,OsakaUniversity,
Ibaraki,Osaka,Japan;GraduateSchoolofPharmaceuticalSciences,OsakaUniversity,
Suita,Osaka,Japan
HIROYUKINOJI (cid:1) DepartmentofAppliedChemistry,GraduateSchoolofEngineering,
TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan
NORIMICHINOMURA (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,Kyoto
University,Kyoto,Kyoto,Japan;ResearchAccelerationProgram,MembraneProtein
CrystallographyProject,JapanScienceandTechnologyAgency,Kyoto,Kyoto,Japan;
ERATO,IwataHumanReceptorCrystallographyProject,JapanScienceandTechnology
Agency,Sakyo-ku,Kyoto,Japan
OSAMUNUREKI (cid:1) DepartmentofBiologicalSciences,GraduateSchoolofScience,
TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan
SATOSHIOGASAWARA (cid:1) DepartmentofCellBiology,GraduateSchoolofMedicine,Kyoto
University,Kyoto,Kyoto,Japan;DepartmentofChemistry,GraduateSchoolofScience,
ChibaUniversity,Chiba,Chiba,Japan