Reviews of Physiology, Biochemistry and Pharmacology

168 Pages·2004·2.184 MB·English

by  H.-J. Apell (auth.)

Reviews of Physiology, Biochemistry and Pharmacology 150
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RevPhysiolBiochemPharmacol(2003)150:1–35
DOI10.1007/s10254-003-0018-9
H.-J.Apell
Structure–function relationship
in P-type ATPases—a biophysical approach
Publishedonline:17June2003
(cid:1)Springer-Verlag2003
Abstract P-typeATPasesarealargefamilyofmembraneproteinsthatperformactiveion
transport across biological membranes. In these proteins the energy-providing ATP hy-
drolysisiscoupledtoion-transportthatbuildsupormaintainstheelectrochemicalpoten-
tialgradientsofoneortwoionspeciesacrossthemembrane.P-typeATPasesarefoundin
virtuallyalleukaryoticcellsandalsoinbacteria,andtheyaretransportersofabroadvari-
etyofions.Sofar,acrystalstructurewithatomicresolutionisavailableonlyforonespe-
cies,theSRCa-ATPase.However,biochemical andbiophysicalstudiesprovide anabun-
dance of details on the function of this class of ion pumps. The aim of this review is to
summarizetheresultsofpreferentiallybiophysicalinvestigationsofthethreebest-studied
ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to
compare functional properties torecent structuralinsights with the aim of contributing to
theunderstandingoftheirstructure–functionrelationship.
Introduction
All living cells are surrounded by membranes that separate their strictly controlled cyto-
plasmic contents from their environment, and within cells numerous compartments with
specific functions and different compositions of components are enclosed also by mem-
branes.Thesemembranesconsistoflipidbilayers,whichareeffectivebarriersformostof
thewater-solublesubstances,suchasions,sugars,andaminoacids.Toperformitsmetab-
olism, a cell needs selective and controlled transport of substrates and of end products of
the metabolic processes across these membranes. This transport function is performed by
membraneproteins.
)
H.-J.Apell( )
DepartmentofBiology,
UniversityofKonstanz,
FachM635,78457Konstanz,Germany
e-mail:[email protected]
2 RevPhysiolBiochemPharmacol(2003)150:1–35
Besidesthe separation of aqueousphases,asecond function ofmembranesisthe stor-
age of energy in the form of chemical potential gradients, Dm ¼RT(cid:2)ln(cid:1)c0=c00(cid:2), of sub-
i i i
stancesiinthecaseofunchargedsubstancesorinthecaseofionsintheformofelectro-
chemicalpotentialgradients,Dm~ ¼RT(cid:2)ln(cid:1)c0=c00(cid:2)þzFðj0(cid:1)j00Þ.
i i i i
Onthebasisofthermodynamicprinciples,twoclassesoftransportproteinscanbedis-
criminated:proteinsthatperformpassiveandactivetransport.Passivetransportisdefined
by facilitated diffusion “downhill” along the (electro-)chemical potential gradient of the
transportedsubstancewherebytheenergygradientdissipates.Activetransportoccurs“up-
hill,” increasing the (electro-) chemical potential of the transported substances. This is
possible only if energy in the form of free energy, DG, is provided from another process
whichiscoupledtothetransportacrossthemembrane.Thisenergyinputhastobelarger
thanthe(electro-)chemicalpotential,jDGjgt;Dm~.Activeion-transportproteinsinanimals
i
are mostly ion transporters, so-called ion pumps. A careful and detailed introduction into
thebiophysicsofionpumpscanbefoundinthemonographElectrogenicIonPumps(L(cid:201)u-
ger1991).
Energy sources that power active ion transport are light, e.g., in bacteriorhodopsin
(Stoeckenius1999;DerandKeszthelyi2001;LanyiandLuecke2001),redoxenergy,e.g.,
inthecytochromecoxidase(Michel1999;Wikstrom2000;Abramsonetal.2001),orde-
carboxylation, e.g., in ion-translocating decarboxylases (Dimroth 1987; Michel 1999;
Wikstrom2000;Abramsonetal.2001).Themostcommonenergy-producingmechanism
is,however,ATPhydrolysisintransportATPases.
Ion-motiveATPasesarethelargestandmostdiverseclassofionpumps.Threegroups
arediscussedintheliterature:(a)F-typeATPases(Dimrothetal.2000;Papaetal.2000;
CapaldiandAggeler2002;Senioretal.2002),whichworkinmanycasesinreversedirec-
tionasso-calledATPsynthetases,e.g.,intheinnermitochondrialmembraneorinthethy-
lakoidmembraneofchloroplasts.(b)V-typeATPases(Szeetal.1992;Nelson1995;For-
gac 1999)whichare ubiquitous H-ATPases withastructure related tothat of F-typeAT-
Pases. They are found in cellular organelles of an ever-increasing number of different
cells.(c)P-typeATPases,whicharefoundinvirtuallyalleukaryoticcellsandalsoinbac-
teria.
P-TypeATPases
In contrast to the other two types of ion-motive ATPases, P-type ATPases are of a much
simplerstructure (Møller etal. 1996).They have an a-subunit of approximately 100kDa
that contains all components essential for enzymatic activity and transport. Examples of
such single-subunit P-type ATPases are, e.g., Ca-ATPases (Carafoli 1992; Lee and East
2001).Na,K-ATPaseandH,K-ATPasearefunctionalonlyifassembledtogetherwithab-
subunit (McDonough et al. 1990; Geering et al. 2000; Geering 2001). In the case of the
Na,K-ATPase,inspecifictissuesag-subunitwasfound,whichisalsodiscussedasaregu-
latory device (Berrebi-Bertran et al. 2001; Cornelius et al. 2001; Therien et al. 2001).
Meanwhile, a whole family of such regulators was identified, called FXYD proteins (Be-
guinetal.2002;Gartyetal.2002).AK-ATPaseofE.coli(theso-calledKdp-ATPase)is
composedofthreedifferentpolypeptides(Epsteinetal.1990;Altendorfetal.1992).
A second fundamental difference between P-type ATPases and the other ion-motive
ATPases is their enzymatic reaction mechanism (Glynn 1985; Lancaster 2002), which
RevPhysiolBiochemPharmacol(2003)150:1–35 3
Fig.1 StructureoftheCa-ATPaseofthesarcoplasmaticreticuluminbothprincipalconformationsasre-
solvedbytheircrystalstructure.Left:InitsconformationCaE (PDBfile1EUL)thespatialresolutionwas
2 1
2.6(cid:13)(Toyoshimaetal.2000).Right:ThestructureintheE conformation(PDBFile1IWO)wasstabilized
2
bytharpsigargin(notshown)andobtainedfromcrystalswitharesolutionof3.1(cid:13).(ToyoshimaandNomu-
ra2002)
containsaphosphorylatedintermediate.ThegphosphateofATPistransferredtoahighly
conservedaspartylresidueinthelargecytoplasmicloopbetweentheforthandfifthtrans-
membrane segment. Specific to P-type ATPases is also that the enzymatic activity (and
consequently ion transport) can be inhibited by ortho-vanadate, which acts as a tightly
boundtransition-stateanalogueofphosphate(Cantleyetal.1977).
P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, where
they actively transport various ions. They are distributed in different classes (I–IV) and
severalsubgroups(SweadnerandDonnet2001)accordingtotheionstheytransport:Na+,
K+,Ca2+,H+,Mg2+,Cu2+,Cd+,Hg+,andevenCl-(Gerencser1996).
Structuralproperties
Althoughtheirmolarmassesvarybetweenabout70and100kDa,thefirstfivetransmem-
branedomainsandthelargecytoplasmicloop,whichformsthemainpartoftheenzymatic
machinery, are well conserved for all P-type ATPases. Yeast proteins mostly have six
transmembrane domains,whilethose from animaltissues preferentiallyhaveten (Swead-
nerandDonnet2001).
A breakthrough in the understanding of structure–function relationships was made
when the first highly-resolved 3D structure of a P-type ATPase became available with a
resolutionof2.6(cid:13)(Fig.1),theCa-ATPaseofthesarcoplasmaticreticuluminitsE con-
1
formation with 2 Ca2+ ions bound (“Ca E ;” Toyoshima et al. 2000). The structure con-
2 1
firms the topological organization of ten transmembrane helices deduced for Ca, Na,K-,
H,K-andH-pumpsbybiochemicaltechniques(MacLennanetal.1985),andthestructure
reveals several unexpected features. It was found (Toyoshima et al. 2000) that (a) both
ionsarelocatedsidebysidewithadistanceof5.7(cid:13)closetothemiddleofthetransmem-
branesectionoftheprotein,(b)theionbindingsitesaresurroundedbythetransmembrane
4 RevPhysiolBiochemPharmacol(2003)150:1–35
helices M4–M6 and M8, (c) the a helices M4 and M6 are partly unwound to provide an
efficientcoordinationgeometryforthetwoCa2+ions,and(d)acavitywitharatherwide
opening, surrounded by M2, M4, and M6 is discussed as an access structure on the cyto-
plasmicside.TheoutletofCa2+islikelytobelocatedintheareasurroundedbyM3–M5.
The details of Ca2+ occlusion sites fit well with that deduced in extensive mutagenesis
studies (Clarke et al. 1989a, MacLennan et al. 1997). The parts of the protein protruding
into the cytoplasm are divided into three domains, two domains, N (nucleotide) and P
(phosphorylation), are formed by the loop between M4 and M5, well separated from a
thirdAdomain(actuatoror anchor)formedbythe loopbetween M2andM3andthetail
leading into M1. The fold of the P-domain is like that of L-2-haloacid dehalogenase and
related proteins with homologies to P-type pumps in conserved cytoplasmic sequences
(Sarasteetal.1990;Aravindetal.1998).
Recently, the structure of the SR Ca-ATPase in its second principle conformation, E ,
2
stabilizedbythespecificinhibitortharpsigargin[“E (TG)”],becameavailablewithareso-
2
lution of 3.1 (cid:13) (Toyoshima and Nomura 2002). Due to the low Ca2+-binding affinity in
theE state,itwasnotpossibletoobtaincrystalswithCa2+ionsboundwhichwouldallow
2
adirectdeterminationof thepositionof thebindingsites.Itwasproposedthat inE (TG)
2
thecounterionsH+areboundandaccesstotheluminalsitesisalreadylocked.Neverthe-
less,bycomparisonofbothcrystallizedforms,Ca E andE (TG),itispossibletodescribe
2 1 2
a number of changes in the protein structure that are important for conclusions on func-
tionalpropertiesrelatedtoenzymaticandtransportactivity.[Thesedifferencesareimpres-
sivelyvisualizedassupplementaryinformationtoToyoshimaandNomura(2002)onNa-
ture’s website (www.nature.com).] The three cytoplasmic domains, N, P, and A, which
form the enzymatic machinery, are wide open in the Ca E form, and they are folded to-
2 1
gether to a much more compactassembly inthe E (TG) form (Fig. 1). Thistransition re-
2
quiresmovementsoftheNdomainofabout50(cid:13)andarotationoftheAdomainofabout
110(cid:7). The cytoplasmic domains move as a whole in a M10-to-M1 direction (Toyoshima
andNomura2002).ThePandNdomainsthemselvesarenotchangedbetweenbothcon-
formations.
Despitethepreviouslyoftendiscussedconceptthatinthemembranedomainsnomajor
structural rearrangements are expected between different conformations of the pump, the
reported changes of position and tilt of the first six transmembrane helices are dramatic.
The transition between Ca E and E (TG) is rather complicated and includes partial un-
2 1 2
windingofa-helices,bendingapartofana-helixbyalmost90(cid:7)(M1),changingtilts(M2-
M5),~90(cid:7)rotations(M6),shiftstowardsthecytoplasmicside(M1,M2)orshiftsinoppo-
sitedirectionby5(cid:13),whichisalmostoneturnofana-helix(M3,M4).(Formoredetails
seeToyoshimaandNomura2002.)Withrespecttothecytoplasmicdomainsoftheenzy-
maticmachinerytheinterplaybetweentheseisobvious,anditisclearlypossibletoimag-
inetheconceptthatCa2+bindinghastotriggerenzymephosphorylation,andthatarelax-
ationofthephosphorylatedform(andreleaseofthenucleotide)subsequentlydisruptsthe
ionbindingsitesasseeninthecrystallizedE (TG)conformation.Thealmostperfectcoor-
2
dinationofbothCa2+ionsinE (Toyoshimaetal.2000)isabolishedinE (TG)byashift
1 2
ofM4andaclockwiserotationofthethreecrucialresiduesonM6outofsiteI(Toyoshi-
maandNomura2002).
Sofar,theSRCa-ATPaseistheonlyP-typeATPasewithsuchadetailedstructuralres-
olution.From other membersof this family onlyimageswith alower resolutionof about
8(cid:13)areavailable(K(cid:224)hlbrandtetal.1998;Scarborough1999;Hebertetal.2000).Acom-
RevPhysiolBiochemPharmacol(2003)150:1–35 5
Fig.2 ReactionschemeforaP-
typeH-ATPase.E andE are
1 2
theconformationsoftheprotein
withion-bindingsitesfacingcy-
toplasmandextracellularmedi-
um,respectively.Certaintransi-
tionsbetweenneighboringstates
oftheproteinmustbekinetically
inhibited(dashedlines)topro-
duceATP-driventransportcycle
(solidlines)thatpumpsH+ions
outofthecell
parison of such images with a similarly resolved SR Ca-ATPase structure (Zhang et al.
1998) indicates that they agree in most of the important structural details. Therefore, a
computerapproachwasusedinwhichtheconservedhomology,especiallyintheATPhy-
drolysis site of the P-type ATPases (Jørgensen et al. 2001), as well as other aligned con-
servedsegments,weremappedontheSRCa-ATPasestructure.Althoughthehomologyis
highestfortheNa,K-ATPase,thisprocedureledtoreasonableresultsalsoforotherP-type
ATPases(SweadnerandDonnet2001).Mostinsertionsanddeletionswerepredictedtobe
at the protein surfaces, and the similarity proposes a shared folding of all tested P-type
ATPases,despitesomeparticularexceptions.
Therefore, the structural features of the SR Ca-ATPase will be used in the following
paragraphs to represent the considerations of structure–function principles of P-type AT-
Pases.
Principlesoftransportfunctions
The eminent importance of the insights into structural details of the SR Ca-ATPase is
pairedwithafunctionalanalysis,whichismostelaboratefortheNa,K-ATPase.Thetrans-
port mechanism found for this ion pump could be generalized for all P-type ATPases.
Since enzymatic and transport functions have to be coupled, the pump mechanism has to
beacomplexprocess.
Theanalysisoftheion-transportprocessinP-typeATPasesrevealedthatatleastthree
categories of reactions have to occur, performed sequentially in forward or backward di-
rection:(a)ionbindingorrelease,(b)ionocclusionordeocclusion,and(c)transitionsbe-
tweenbothprincipalconformationsinwhichthebindingsitesbecomeaccessiblefromthe
cytoplasm (E ) or from the opposite aqueous compartment (E ). Taking these reactions
1 2
into account, a general reaction scheme can be constructed which has eight states in the
simplestcaseofaH-ATPasethattransfersoneH+perhydrolyzedATP(Fig.2).Ifalltran-
sitions were allowed, such a protein would short-circuit the membrane for H+ ions in the
fashion of an ion carrier, and it would be able to dissipate the energy provided by ATP
hydrolysis without ion transport. Therefore, a number of transitions have to be inhibited
kinetically by the pump protein to perform active ion transport as indicated in Fig. 2 by
dashedlines(L(cid:201)uger1991).Indeed,thisreactionschemewasfoundtorepresentperfectly
thefunctionofaP-typeH-ATPasefromEnterococcushirae(ApellandSolioz1990).The
transportofcounterions,asfoundinmostoftheotherP-typeATPases,canbeconstructed