Molecular Assembly in Natural and Engineered Systems

403 Pages·2011·14.948 MB·1-401\403

by  Stefan Howorka (Eds.)

AcademicPressisanimprintofElsevier
32JamestownRoad, London,NW17BY,UK
Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands
225WymanStreet,Waltham,MA02451,USA
525BStreet,Suite1900,SanDiego,CA92101-4495,USA
(cid:2)
Thisbookisprintedonacid-freepaper.
Copyright(cid:1)2011,ElsevierInc.AllRightsReserved
Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinany
formorbyanymeanselectronic,mechanical,photocopying,recordingorotherwisewithoutthe
priorwrittenpermissionofthePublisher
PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRights
DepartmentinOxford,UK:phone[phone];fax[phone];
email:[e-mail]tyourrequestonlineby
visitingtheElsevierwebsiteathttp://elsevier.com/locate/permissions,andselecting
ObtainingpermissiontouseElseviermaterial
Notice
Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersonsor
propertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseoroperation
ofanymethods,products,instructionsorideascontainedinthematerialherein.Becauseofrapid
advancesinthemedicalsciences,inparticular,independentverificationofdiagnosesanddrug
dosagesshouldbemade
LibraryofCongressCataloging-in-PublicationData
AcatalogrecordforthisbookisavailablefromtheLibraryofCongress
BritishLibraryCataloguinginPublicationData
AcataloguerecordforthisbookisavailablefromtheBritishLibrary
ISBN:978-0-12-415906-8
ISSN:1877-1173
ForinformationonallAcademicPresspublications
visitourwebsiteatelsevierdirect.com
PrintedandBoundintheUSA
11 12 13 14 10 9 8 7 6 5 4 3 2 1
Contributors
Numbersinparenthesesindicatethepagesonwhichtheauthors’contributionsbegin.
Elizabeth H.C. Bromley, Department of Physics, Durham University,
Durham,UnitedKingdom(231)
KevinJ.Channon,DepartmentofPhysics,CavendishLaboratory,University
ofCambridge,Cambridge,UnitedKingdom(231)
Michel T. Dedeo, Department of Chemistry, University of California; and
Materials Sciences Division, Lawrence Berkeley National Laboratories,
Berkeley,California,USA(353)
Eva M. Egelseer, Department of NanoBiotechnology, University of Natural
ResourcesandLifeSciences,Vienna,Austria(277)
Daniel T. Finley, Department of Chemistry, University of California; and
Materials Sciences Division, Lawrence Berkeley National Laboratories,
Berkeley,California,USA(353)
MatthewB.Francis,DepartmentofChemistry,UniversityofCalifornia;and
Materials Sciences Division, Lawrence Berkeley National Laboratories,
Berkeley,California,USA(353)
Christine M. Horejs, Department of NanoBiotechnology, University of
NaturalResourcesandLifeSciences,Vienna,Austria(277)
Stefan Howorka, Department of Chemistry, Institute of Structural and
MolecularBiology,UniversityCollegeLondon,London,UnitedKingdom(73)
MartinHumenik,LehrstuhlBiomaterialien,Universita¨tBayreuth,Bayreuth,
Germany(131)
Nicola Ilk, Department of NanoBiotechnology, University of Natural
ResourcesandLifeSciences,Vienna,Austria(277)
Walter Keller, Institute of Molecular Biosciences, Structural Biology,
UniversityofGraz,Graz,Austria(73)
Tea Pavkov-Keller, Institute of Molecular Biosciences, Structural Biology,
UniversityofGraz,Graz,Austria(73)
Dietmar Pum, Department of NanoBiotechnology, University of Natural
ResourcesandLifeSciences,Vienna,Austria(277)
Han Remaut, Structural & Molecular Microbiology, VIB/Vrije Universiteit
Brussel,Brussels,Belgium(21)
ThomasScheibel,LehrstuhlBiomaterialien,Universita¨tBayreuth,Bayreuth,
Germany(131)
Georg E. Schulz, Institut fu¨r Organische Chemie und Biochemie,
Albert-Ludwigs-Universita¨t,FreiburgimBreisgau,Germany(187)
ix
x contributors
Bernhard Schuster, Department of NanoBiotechnology, University of
NaturalResourcesandLifeSciences,Vienna,Austria(277)
Uwe B. Sleytr, Department of NanoBiotechnology, University of Natural
ResourcesandLifeSciences,Vienna,Austria(277)
Andrew Smith, Lehrstuhl Biomaterialien, Universita¨t Bayreuth, Bayreuth,
Germany(131)
Sophia J. Tsai, UCLA Department of Chemistry and Biochemistry,
UCLA-DOE Institute for Genomics and Proteomics, Los Angeles,
California,USA(1)
Rupert Tscheliessnig, Department of NanoBiotechnology, University of
NaturalResourcesandLifeSciences,Vienna,Austria(277)
Nani Van Gerven, Structural & Molecular Microbiology, VIB/Vrije
UniversiteitBrussel,Brussels,Belgium(21)
Gabriel Waksman, Institute of Structuraland Molecular Biology, University
CollegeLondonandBirkbeckCollege,London,UnitedKingdom(21)
Todd O. Yeates, UCLA Department of Chemistry and Biochemistry,
UCLA-DOE Institute for Genomics and Proteomics, Los Angeles,
California,USA(1)
Preface
The topic of this book—regular protein assemblies—is highly interdisci-
plinary, rapidly expanding, and of great interest for several reasons. Natural
protein assemblies are essential components in every virus or biological cell.
They act as cytoskeletal scaffold, storage containers, or molecular engines for
directionaltransport,cellcontraction,andlocomotion.Proteinassembliesare
also of interest due to their sophisticated molecular architecture. While diffi-
culttoanalyze,theirstructuralcomplexityisincreasinglyelucidatedinatomis-
ticdetails.Moreover,theself-organizationofindividualmolecularentitiesinto
higher-order systems is—from the biophysical perspective—an appealing yet
not fully understood phenomenon.Further,theinspirationbyNature hasled
researchers to rationally design synthetic protein- and peptide-based higher-
order structures that expand the scope of biology. Finally, natural and engi-
neered protein assemblies can be exploited in biomedical or nonbiological
applications. For example, natural systems are already important vaccine tar-
gets, while engineered structures are being increasingly utilized in nanobio-
technology for drug delivery. In either case, the exploitation of the self-
assemblingproteinstakesadvantageoftheirrepetitivenatureandthedefined
morphologicalshape.Thesebuilt-incharacteristicscanoptionallybecombined
withnewfunctionalmodulestogeneratebiomaterialsofdesignedproperties.
MolecularAssemblyinNaturalandEngineeredSystemsisoneofthefirst
books to capture this divergent field in its full breadth and depth. The book
brings together different disciplines such as structural biology, protein engi-
neering, and materials science to provide a comprehensive account of the
multifacetedresearchactivities.
Three main aspects are covered. First, the book summarizes recent prog-
ressinthestructuralanalysisofnaturalproteinassemblieswithmorphologies
ofspheres,fibersandsheets.
Spherical bacterial microcompartments: The chapter of Tsai and Yeates
describes thestructureandbiologyofthe metabolic compartments thatcarry
outspecificreactioncascadesintheirinteriors.
Elongated bacterial pili and flagella: Van Gerven, Waksman, and Remaut
offer insight into the biology and structure of adhesion pili and secretion
systems which are key players in pathogenic processes such as recognition
andcolonizationoftargetsurfaces,biofilmformation,andsignalingevents.
xi
xii preface
Sheet-like bacterial S-layers: Pavkov-Keller, Howorka, and Keller review
the structure of S-layers. These protein lattices cover the cell wall of many
biotechnologically relevant or pathogenic bacteria and occur on almost all
archaeawheretheyconstitutetheonlycellwallcomponent.
Thefibersoftheremarkablytoughyetlight-weightdraglinespidersilkalso
belongtoorderedproteinsystems.Thespidroinspiderproteinsassembleinto
parallel aligned crystallites which are surrounded by elastic nonordered
regions. Humenik, Scheibel, and Smith examine the structural and biological
determinantsatthisintriguinginterfacebetweenstructuralorderanddisorder.
Representing the second aspect, the book demonstrates how structural
understanding has inspired the creation of engineered protein or peptide-
basedassemblies.
Followingtheconceptofstructure-basedintervention,Schulzexplainshow
otherwise monomeric proteins can be engineered to form homo-oligomers of
definedarchitecture.
In a complementary chapter, Bromley and Channon show that alpha-
helical peptide assemblies can be rationally designed to add new function to
thebottom-upstructuresusefulforapplicationsincellbiology.
Thelasttopicofthebookemphasizeshownaturalandengineeredsystems
canbeexploitedforawiderangeofapplicationsincludingbiocatalysis,biode-
livery,ormaterialsscience,tonameafew.
Sleytr,Schuster,Egelseer,Pum,Horejs,Tscheliessnig,andIlkdemonstrate
that S-layer proteins are modular building blocks for the production of new
supramolecularmaterialsandnanoscaledeviceswhichcanbeusedinmolecu-
larnanotechnology,nanobiotechnology,biomimetics,andsyntheticbiology.
Newmaterialscanalsobegeneratedusingviralcapsids,whichasshownby
Dedeo, Finley, and Francis, can be exploited as self-assembling templates to
assemblechromophoresforuseinlightharvestingandphotocatalyticapplica-
tions,orasdeliveryvehiclesfordrugandimagingcargo.
Severalaspectsofbiotechnologicalapplicationsarealsocoveredinmostof
thepreviouschaptersaboutnaturalandengineeredassemblysystems.
The book aims to reach a broad audience of researchers in structural
biology, molecular biology, bionanotechnology, biochemistry, biophysics, and
relatedareas,aswellasadvancedundergraduateandgraduatestudents.
I hope that you enjoy reading this book which demonstrates that protein
assembliesareahighlyinterdisciplinaryfieldwhich—guidedbyexcitingstruc-
tural biology—spans the nano- and microscale and can find many bio-related
applicationswhiletranscendingintononbiologicalareas.
StefanHoworka
London,August2011
Bacterial Microcompartments:
Insights into the Structure,
Mechanism, and Engineering
Applications
SophiaJ.Tsaiand
ToddO.Yeates
UCLADepartmentofChemistryand
Biochemistry,UCLA-DOEInstitutefor
GenomicsandProteomics,LosAngeles,
California,USA
I.Introduction.................................................................................... 1
II.BacterialMicrocompartmentFormandFunction..................................... 2
III.ShellProteins:StructuresandMechanisms............................................. 5
IV.Higher-LevelOrganization.................................................................. 9
V.FutureDirectionsforResearchandDesignApplications........................... 11
VI.ClosingRemarks.............................................................................. 16
References...................................................................................... 16
Bacterialmicrocompartmentsarelargesupramolecularassemblies,resembling
virusesinsizeandshape,foundinsidemanybacterialcells.Aprotein-basedshell
encapsulatesaseriesofsequentiallyactingenzymesinordertosequestercertain
sensitivemetabolicprocesseswithinthecell.Crystalstructuresoftheindividual
shellproteinshaverevealeddetailsabouthowtheyself-assembleandhowpores
through their centers facilitate molecular transport into and out of the micro-
compartments. Biochemical and genetic studies have shown that enzymes are
directed to the interior in some cases by special targeting sequences in their
termini. Together, these findings open up prospects for engineering bacterial
microcompartments with novel functionalities for applications ranging from
metabolicengineeringtotargeteddrugdelivery.
I. Introduction
Bacterialmicrocompartments(BMCs)arelarge,protein-basedassemblies
presentinsidemanybacterialcells.1Theywerefirstvisualizedinthelate1950s
and1960sinsidecyanobacteriaaselectrondense,polyhedrallyshapedbodies,
reminiscent of viruses or phage capsids.2,3 They are now recognized to be
metabolic compartments that carry out specific series of reactions in their
interiors.4–6VarioustypesofBMCssequesterdifferentmetabolicpathwaysin
ProgressinMolecularBiology Copyright2011,ElsevierInc.
andTranslationalScience,Vol.103 1 Allrightsreserved.
DOI:10.1016/B978-0-12-415906-8.00008-X 1877-1173/11$35.00
2 TSAI AND YEATES
different bacterial lineages. These different types of BMCs are unified by
having an outer shell assembled from protein subunits that are homologous
(i.e., evolutionarily related) between the various systems.7 The three-dimen-
sional structures of numerous shell proteins have recently been elucidated,
providing considerable insight into questions of architecture, biochemical
mechanisms, and evolutionary relationships.7–17 Together with biochemical
andbioinformaticstudies,thisstructuralunderstandinghasopenedupexciting
prospectsfordesigningBMCswithnovelproperties,andforusingBMCsasa
broadframeworkforsystemsbiologyresearchandapplications.
II. Bacterial Microcompartment Form and Function
BMCs vary considerably in size and shape (Fig. 1). The smallest BMCs
knownarethoseofthealpha-typecarboxysome.InHalothiobacillusneapolita-
nus, diameters of their alpha-carboxysomes have been reported to be in the
range80–120nm.18,19BMCsofthistypearealsothemostgeometricallyregular
amongthosethathavebeenvisualizedbyelectronmicroscopy.Recentelectron
cryo-tomographystudieshaveshownthatalpha-carboxysomeBMCsarenearly
icosahedralinshape;20roughlyflattriangularfacetscometogetherat30edges
and12pentagonalvertices.18,20Accordingtothesedimensions,BMCshellsare
composedofatleastafewthousandshellproteinsubunits.13,15,21Itisnotable
that,evenforthesemostregularBMCs,theirsizesvarywithinagivencell,with
anindividualcelltypicallycontainingbetweenafewandseveralBMCs.Other
types of BMCs (besides carboxysomes) generally appear to be less regular,
A B C D
C
S
FIG.1. Visualizationofbacterialmicrocompartments.(A)Transmissionelectronmicrograph
of thin-sectioned H. neapolitanus cells showing alpha-type carboxysomes (scale bar¼100 nm).
(B)PurifiedcarboxysomesfromH.neapolitanus(scalebar¼100nm)(S¼shell;C¼carboxysome).
PanelsAandBarecourtesyofGordonCannonandSabineHeinhorst.(C)beta-typecarboxysomes
fromSynechocystisPCC6803areshowninthinsection(scalebar¼50nm)(courtesyofRobbie
Roberson and Allison van de Meene). (D) Isolated pdu microcompartments from Salmonella
enterica spp. Typhimurium LT2, visualized by negative stain (scale bar¼100 nm) (courtesy of
TomBobik).FigureandlegendfromRef.17.
BACTERIALMICROCOMPARTMENTS 3
typicallyretainingsomepolyhedralcharacter,suchasrecognizableedges,but
failingtoconformtoicosahedralshapeandsymmetry.22–24Structuralpolymor-
phismappearstobeacommonfeatureofmosttypesofBMCs.
ThecellularfunctionsofBMCsaretoencapsulateaseriesofsequentially
actingenzymesandthemetabolicintermediatesinvolvedinthepathwaysthey
catalyze (Fig. 2). Several distinct types of BMCs are recognized, with the
carboxysome being the founding member.17,25 Carboxysomes are present in
all cyanobacteria, and some chemoautotrophs—bacteria that fix inorganic
carbon (i.e., CO ) into organic form.5,26 Carboxysomes encapsulate two
2
enzymes:carbonicanhydrase(CA)andRuBisCO.19Carbonicanhydrasedehy-
dratesbicarbonatetoproduceCO ,afterwhichRuBisCOcombinesCO with
2 2
the five-carbon molecule 1,6-ribulose bisphosphate (RuBP) to produce two
molecules of the three-carbon 3-phosphoglycerate (3-PGA). Colocalization of
the two enzymes together is believed to supply the notoriously inefficient
RuBisCO enzyme with a high concentration of its CO substrate.4,27 Based
2
on the overall reaction scheme, bicarbonate and RuBP must cross the outer
proteinshellintothecarboxysomeinterior,and3-PGAmustexit.Whilethese
substrates and products must be able to pass easily across the shell, the CO
2
producedinsidethecarboxysomemustbefixedbyRuBisCObeforeitescapes.
Structuraldatainthepastfewyearshasadvancedtheideathattheproteinshell
plays a key role in controlling transport into and out of the carboxysome and
otherBMCs,butnumerousquestionsremain.
Experiments have been conducted on two other types of BMCs that are
very different from the carboxysome but are similar to each other in key
respects. The propanediol utilizing (Pdu) and ethanolamine utilizing (Eut)
BMCs metabolize the three-carbon 1,2-propanediol (1,2-PD) molecule and
the two-carbon ethanolamine molecule, respectively23,28–30 (Fig. 2). These
BMCsarewidelydistributedacrossthebacterialkingdom,includinginenteric
bacteria found in human hosts. Both Pdu and Eut microcompartments are
presentinSalmonellaenterica,andtheEutoperonispresentinsomestrainsof
E. coli. The metabolic pathways for degrading propanediol and ethanolamine
both go through aldehyde intermediates, propionaldehyde and acetaldehyde,
respectively. Aldehydes are chemically reactive, and experiments have shown
that containing them within the BMC so they can be reactedupon in further
enzymaticstepsavoidstheotherwisecytotoxiceffectsofhighconcentrationsof
aldehyde.31–33InthecaseoftheEutBMC,analternativeargumenthasbeen
put forth based on data showing that the key defect when ethanolamine is
metabolized outsideof aBMC isevaporative loss ofthevolatileacetaldehyde
fromthecell.34Ineithercase,theprincipleisthatasmallmoleculeneedstobe
metabolized in multiple steps without allowing escape of an intermediate
species. This general idea applies as well to the carboxysome, where the
intermediate that needs to be further metabolized prior to escape is CO .
2
FIG.2. Geneorganizationandproposedmetabolicpathwaysfortwotypesofbacterialmicro-
compartments(BMCs).Genesarecoloredtoindicatetheirhomology.AllBMCshellproteinsare
light blue. For each microcompartment, the key sequestered intermediate is boxed in orange.
(A) Function of the carboxysome in enhancing CO fixation. Gene organizations for alpha and
2
beta-typecarboxysomesareshownbelow.(B)Acurrentmodelforthefunctionofthepropanediol
utilization(Pdu)microcompartmentinmetabolizing1,2-propanediol.Thegeneorganizationfor
thepduoperonisshownbelow.FigureandlegendadaptedfromRef.1.
BACTERIALMICROCOMPARTMENTS 5
ThePduandEutBMCsstandapartfromthecarboxysome,however,interms
ofthecomplexityoftheirmetabolicenzymesandpathways.InthecaseofPdu,
asmanyaseightdistinctenzymesubunitsarepresentcarryingoutfivedifferent
reaction steps.Furthermore,anumberofbulkycofactors arerequiredbythe
encapsulated enzymes. A firm understanding of which reactions occur inside
the Pdu BMC is still incomplete, but it is likely that cobalamin (B ), coen-
12
zyme-A,NADH,iron–sulfurclusters,andpossiblyATParerequired.6Someof
these turn over or degrade during activity, leading to important questions
regarding how such bulky molecules might be able to enter and exit the
BMC, while the small molecule intermediate (i.e., propionaldehyde) fails to
escape.Structuraldatahaveprovidedsomecluestothispuzzle.8,11,14
TheshellsofBMCsareformedmainlybyproteinsfromtheBMCfamily,that
is,proteinsthatbearoneormoreBMCdomainsapproximately100aminoacids
long.7,21Afewthousandofthesesmallproteinsself-assembletoformtheouter
polyhedralstructure(Fig.3).ThesequencesofBMCshellproteinswerefirst
identifiedfrompurifiedcarboxysomes.19BMCshellproteinswerethenshown
bysequencehomologytobewidelydistributedacrossthecyanobacteria,often
arrangedonthebacterialchromosomeinproximitytootherproteinsinvolvedin
carboxysome function, including RuBisCO and carbonic anhydrase.26,35 In
1994, proteins from the BMC family were found in Salmonella, an enteric
bacterium, in the large multigene pdu operon that encodes enzymes for 1,2-
PDmetabolism.28ThepresenceofBMCproteinsinthemidstofenzymesfor
1,2-PD metabolism suggested that this pathway was compartmentalized in
Salmonella,whichwasthenestablishedexperimentally.23ThePdumicrocom-
partmentwasshowntohavearoleinpreventingaldehydetoxicityinthecytosol,
asnotedabove.Aprotocolwasdevelopedforobtaininghomogeneousprepara-
tionsofPdumicrocompartments,allowingacompositionalanalysisofthisBMC,
whosecontentsareconsiderablymorecomplexthanthecarboxysome.31
III. Shell Proteins: Structures and Mechanisms
Using modern sequence searching algorithms, some 2000 distinct BMC
shell proteins can be found distributed across at least 10 different bacterial
phyla;they appear tobeabsentfromeukaryotes andarchaea.Theirscattered
distribution suggests that BMC shell proteins have been spread by horizontal
genetransfer,sometimesbetweenhighlydivergentbacterialspecies.Basedon
genomicanalysisofenzymesencodedinthevicinityofBMCshellproteinsin
different bacteria, it has been surmised that there are probably about seven
different metabolic categories of BMCs17,22,25; only the carboxysome and the
Pdu and Eut types have been studied biochemically or genetically,
though BMC shell proteins from others have been elucidated structurally.16