Comprehensive biophysics. / Volume 9, Simulation and modeling

758 Pages·2012·75.042 MB·English

by  Weinstein, Harel

1.1 Volume Introduction
HJ Dyson,The Scripps Research Institute, LaJolla, CA,USA
r2012ElsevierB.V.Allrightsreserved.
We are entering a new age of unprecedented improvement in picosecondstodays.Cross-fertilizationbetweensolutionand
thetechniquesavailableforthebiophysicalcharacterizationof solid-stateNMRhasledtohappyinnovationssuchastheuse
biological molecules. The impetus derived from the major ofresidualdipolarcouplingstoaugmentNOEandJ-coupling
biological and biomedical mega-projects that have driven re- informationforstructuredeterminationandtheemployment
search in the last 10 years or so not only has given rise to ofmultidimensional techniques insolid-state NMRtoassign
profoundimprovementsintechniquesthathavebeenaround spectraofuniformlylabeledmolecules.
sincethe1940sand1950s,butalsohasseenthedevelopment Spectroscopicmethodsingeneralhaveundergonemassive
of completely new methods designed to probe and measure changes in the last 10 years. Electron spectroscopies have
characteristics of biomolecules that were barely known or multiplied, giving rise to a bewildering variety of acronyms
hintedatintheearlydays.Aswell,wemaytakegreatpridein and a great deal of exciting new data. The traditional spec-
the fact that the advances in biophysical and biochemical troscopies, UV-visible, CD, and Raman, continue to provide
techniques have in many cases driven and outpaced the de- theessentialbaselinedata,buteventheyarebeinggussiedup
velopments made with reference to simpler chemistries: be- intheirnewincarnations,withtwo-dimensionalapplications
cause the study of biological molecules is so much more and extensive data analysis. Two methods stand out for
challenging thanthatofsmall molecules, a concomitant level startling innovations in the last few years. Who would have
ofingenuityhasbeennecessarytoobtaininformationonthem. thought that we would now routinely see applications of
And whatinformation wecan nowcall upon! The steady single-molecule fluorescence spectroscopy, and that these ap-
development of the structural techniques X-ray crystal- plicationswouldprovidesomuchunprecedentedinformation
lography, NMR, and electron microscopy now allows us to and give rise to so many new ideas. We can only anticipate
image cellular organelles and machines at or near atomic that these and similar methods will continue to develop in
resolution. We have seen with amazement and delight the equallystartlingways.
three-dimensionalstructuresofribosomes,nucleosomes,and Afinalnoteontheoverallapproachthatisnowbecoming
proteasomes. Viruses of many types can be imaged by X-ray characteristicofbiophysics.Thefieldisincreasinglyembracing
crystallography and electron microscopy. Membrane protein theso-called‘high-throughput’methodologies.Youwillseea
crystal structures have begun to appear. These structural in- marked emphasis on this aspect in many of the chapters in
sights provide the basis for imagining the workings of the this volume, from the description of high-throughput meth-
living cell – and, because it is unlikely that an entity as het- odsofproteinsynthesis,throughroboticcrystallizationtrials
erogeneous as a cell will ever itself be crystallized, we have with arrays of conditions employed to maximize the likeli-
manyothertoolsinourrepertoirethatcanbeusedtofurther hoodthatamoleculeofinterestwillcrystallize,torapidcrystal
illuminatetheproblem. screening,datacollection,anddataanalysis.Oneofthemajor
AlthoughNMRhastraditionallybeenthe‘littlebrother’in tools in the high-throughput world is mass spectrometry,
thebiomoleculefield,thesizelimitsonstructuralstudiesare which hasdevelopedinto ahighlyspecializedand extremely
becoming less of a limitation and more of an incentive for precise analytical tool that is now widely employed in just
NMRspectroscopiststoacquirebiophysicalinformationother abouteverybiochemistrylab.Weareclearlypoisedtoobtain
than structures. We are now able to quantitate motions of vast amounts of biophysical data. It is incumbent on us to
moleculesandtocorrelatethemotionswithcatalyticreactions makesurethatthesedataareproperlyorganizedandremain
and protein folding. Innovations in design of spectrometer accessibletothegeneralscientificpublic.
hardwareandsoftware,isotopiclabeling,andpulsesequences Finally,thelastfrontier,theuseofcomputationtopredict
nowallow us to study thedynamics of molecular complexes and verify biophysical quantities (Volume 9), is also a field
andmachinesofunprecedentedsizeinsolutionbyNMR,and where unprecedented progress has been made in the last 10
enormous advances in solid-state NMR techniques promise yearsorso.Partoftheadvancehasbeeninthesizeandpower
high-resolution structural data where once only broad dis- oftheavailablecomputingmachinesandtheingenuityofthe
persion spectra were possible. It is awe-inspiring to contem- practitionersinemployingparallelandefficientalgorithmsto
plate the progress that we have seen in the last 10 years. My enhancethepotentialofthehardware.Yetitisclearthat‘‘we
good friend Gitte Vold, whose untimely death in 1999 arenotthereyet.’’Aprioripredictionofbiophysicalattributes
shocked and saddened us all, would be amazed (and de- of biomolecules remains a challenge. Where the quantities
lighted) to see the range of information now available from measured are limited in complexity, it maybe quite possible
her beloved solid-state NMR experiments. Not only can we to describe them theoretically and thus to predict the out-
obtain high-resolution structural data from the magic-angle comes of experiments. However, one of the hallmarks of
spinningexperimentsthatwerejustbeginningtobeemployed biological systems is a satisfying degree of complexity, even
forbiomoleculesattheturnofthetwenty-firstcentury,butthe unpredictability. In spite of the progress made by our com-
whole range of relaxation measurements pioneered by Gitte puting friends, I do not think that our job as experimental
have been extended into the solution state to give expansive biophysicistsisoveryet.
picturesofthedynamicsofbiomoleculesontimescalesfrom WelcometoVolume1oftheComprehensiveBiophysicstext.
ComprehensiveBiophysics,Volume1 doi:10.1016/B978-0-12-374920-8.00101-6 1
2.1 Volume Introduction
PSchwille, Biophysics– Schwille Lab,Dresden, Germany
r2012ElsevierB.V.Allrightsreserved.
Abbreviations FRET Fo¨rsterresonanceenergytransfer
CCD charge-coupleddevice
The cell is the fundamental unit of life, and the quest for its matches the relevant timescales of fundamental biological
understanding has shaped the discipline of biologysince the processes.Forthisreason,ourvolumeonthebiophysicalin-
earlydaysofopticalinstruments,ofLeeuwenhoekandHooke vestigations of cells is divided into two main sections, in
in the sixteenth century. It is fair to state that only since the structuralanddynamicinvestigations.
adventofthemicroscope,atfirstnotmuchmorethanasingle We first address the state of the art of displaying cellular
carefully crafted lens, have researchers begun their journey structures, down to the level of single molecular units. Here,
intotheuniverseoflivingmatter,whichsoonappearedtobe we have faced an enormous development during the past
more complex and incomprehensible than one could have decades,whichstartedwiththeintroductionandwidespread
everexpected.Today,severalhundredyearslater,wehaveac- use of the scanning confocal microscope. But also scanning
quired a large body of knowledge about the minimal func- probe microscopy, one of the key technologies to access
tionalelementsoflife,andbeguntounderstandtheirintricate atomic and molecular dimensions on surfaces, thus being a
relationships, their organization in large and immensely hallmarkofnanotechnology,haslongleftitsphysicalcomfort
complex reaction networks. We have acknowledged the im- zone of vacuum and low temperature, and atomic force mi-
portance of self-assembly, self-organization, and self-repli- croscopyonlivecellsinwaterorevenphysiologicalbufferhas
cation, and the essential concepts of compartmentalization becomereality.Thesamedevelopmentcouldbeseenforop-
andmodularityinlivingentities.Wearestillnotattheendof tical near-field microscopy. X-ray microscopy has been
this long journey, and far from understanding life in all its brought to the level where subcellular organization can be
breathtaking details, but we have come a long way. Biology visualized. Electron microscopy, through the technical ad-
and life sciences in general have become the most vibrant, vancesofcryopreparationandtomography,hasarrivedatthe
powerful,andfastestgrowingdisciplinesinacademicresearch, resolution of large protein complexes and extremely well-
and their traditional boundaries to chemistry, physics, and preserved cellular ultrastructures with dimensions far below
even the engineering subjects have begun to dissolve. All of the fundamental barrier of optical diffraction. Finally, this
this is the consequence, but also the reason, for the ever in- barrieritself,asofarunbeatablelimitinfar-fieldmicroscopy,
creasingrelevanceoftechnologyinthestudyoflivingmatter. has been challenged lately by the development of a set of
WhileHookestillhadtousesunlightorcandlesfortheillu- fluorescence-based technologies, and finally been overcome
mination of his biological samples, and to draw by hand all by utilizing the parameter of time, that is, the possibility to
thefascinatingstructureshesawwithhisrudimentary(yetin separately address and switch single fluorescent emitters on
terms of magnification already quite powerful) microscopes, and off for data collection. The parameter of fluorescence
we nowadays enjoy all the comfort that modern physics-dri- lifetime, and the widely applied phenomenon of Fo¨rster res-
ventechnologyprovidesus,suchaslasersinallspectralran- onance energy transfer (FRET) have helped tremendously in
ges, and high-speed charge-coupled device (CCD) cameras accessingeventhenanometerscaleinresolvingdistancesbe-
with single molecule sensitivity. Today, optical microscopy tweenmoleculesandmolecularmodules.Label-freeanalytical
with its manifold of variants and technological advances is microscopy has also made a big step forward, through the
still one of the major driving forces in understanding bio- introductionofCoherentAnti-StokesRamanScattering,which
logicalsystems,althoughitisincreasinglycomplementedbya selectivelyallowstheexcitementofspecificvibrationsinbio-
large body of technically more demanding methods, such as molecules, and has provedto be a very promising technique
atomicforce,electron,andX-raymicroscopy. particularly for the imaging of lipidic structures, such as
The introduction of fluorescence to the biosciences, par- membranes.
ticularlyintheformofbrightandphotostableextrinsicprobes The second major part of this volume is devoted to
that can be selectively and specifically attached to biomole- methods that address cellular and molecular dynamics on
cules,mustcertainlybeunderstood asamajor breakthrough timescales from minutes down to microseconds. Here in
in the imaging of cellular structures and ultrastructures. particular, fluorescence technology has been vital, and the
Fluorescence microscopy has opened up a new vista of dis- possibilitytodisplayandresolvesinglemoleculefluorescence
playinglife,notonlyintheestheticsensethatitbroughtcolor is central to manyof these techniques. Already in the 1990s,
into aworld of black and white, allowing us to generate im- researchers had accomplished the task of tracking single bio-
agesofstunningbeautytodecoratebookcoversandhallways molecules. The first experiments were carried out on model
of life science institutes, but also in the much more relevant membranes, but through the introduction of better CCD
way that it opened the window much more widely to better technology and several other technical advances, we are
access the dimension of time, with a resolution that now nowadaysabletotracksinglemoleculesinthelivingcelland
ComprehensiveBiophysics,Volume2 doi:10.1016/B978-0-12-374920-8.00201-0 1
2 Volume Introduction
even organism. Also quite early, researchers began to draw biophysical technology development have shared their ex-
informationaboutdynamicprocessesincellsbyinvestigating perienceanddisplayedthecurrentcuttingedgeoftheirtech-
therapidlyshiftingspecklepatternsinimagesobservedatlow nology,whichwillofcoursefaceconstantfurtherexpansion.
density labeling of cellular structures, a technique which is What will the future bring? We can only speculate. To ex-
knownas‘specklemicroscopy’andhasfoundwidespreaduse trapolatefromtheexcitingdevelopmentsofthepastyears,we
incellbiology.Fluorescencecorrelationandcross-correlation will certainly strive for an even better combination of struc-
spectroscopy, spectroscopic techniques carried out in a fixed turalanddynamicanalysis,tobeabletoobservesinglemol-
confocalvolumeelementhavethroughthecombinationwith ecules perform their functional tasks in real time, within
scanning microscopy, made their way into the cell and or- structuresandorganelleswhicharepresentlystillblurreddue
ganism, which enables us now to address concentrations, to their small sizes. Correlative microscopy combining light
mobility coefficients, and reaction rate constants with un- and electron microscopy will deliver ultra-high resolution
precedentedresolution,andopensuptheperspectiveofinsitu images and movies of three-dimensional structures. The
biochemistry in real time. For slower cellular processes in- multiplexing ability opened through the use of fluorescence
volving the reorganization of structures larger than single wavelength and lifetime will be instrumental in addressing
molecules, image correlation has been introduced as a very and understanding biological phenomena on the systems
valuablecomplementarytechnique. level, an approach which is growing strongly in the life sci-
Webelievethatwehaveassembledarathercomprehensive ences.Butthemanyhigh-resolutiontechniquesmayalsoopen
setofchaptersandmethods,displayingthecurrentstateofthe upnewperspectivesforbacterialcellbiology,adisciplinethat
art in the resolution of cellular structures and dynamics, al- hassofarsufferedfromthelackofhigh-qualityimagingdue
thoughthereareofcoursemanymorebiophysicalapproaches to the small cell sizes, but which may be vital to better
out there, which could not be included for the sake of time understandarchetypicalprocessesinsimplersystemsthanthe
and space, and because also a comprehensive volume has to eukaryoticcell.Wewishyouanenjoyableandmuchinspired
be closed at some point. Leading researchers in the field of reading!
4.1 Introduction
YEGoldman andEMOstap,University ofPennsylvania, Philadelphia, PA,USA
r2012ElsevierB.V.Allrightsreserved.
References 1
This volume presents the biophysical investigations of struc- and evolutionary relationships have been extensively re-
ture and function of a large class of energy transducing viewed6–9. Aspects of the structural changes upon ATP hy-
macromoleculesthatarerelatedtoeachotherbytheirability drolysis and downstream effector motions are likely to be
totravelalongtracksofcytoskeletalfilamentsornucleicacids. common among many of these proteins, causing discoveries
Studies of the three classical types of molecular motors, my- in any of them to be of broader interest for understanding
osin, dynein and kinesinwerelargelyseparate fields through othermechano-enzymes.
the early 1990s. Until this time, much of the progress on Inthisvolumewehaveassembledchaptersbymanyofthe
understandingthebiophysicsofmolecularmotorswasmade leadinginvestigatorsonstructure,dynamicsandregulationfor
using muscle as the model system. However, the advent of the molecular motors myosin, dynein and kinesin, their
PCRand sequencing resulted in thediscoveryof large motor cytoskeletal tracks, actin and microtubules, nucleic acid pro-
familiesandcytoskeletalfilamentbindingproteins,providing cessing enzymes, the ribosome, and the portal motors that
investigators with a wealth of new mechanisms and inter- pack DNA or RNA into viral capsids. The F1 ATP synthase
actions to study1–4. Manyof these newlydiscovered proteins whichgeneratesATPinmitochondriawouldalsofitintothis
have kinetic and structural properties that greatly facilitated category of cellular machines, but it is covered in volume 8
structure-functioninvestigations.Additionally,thediversityin withothercellularenergetictopics.TheGTP-bindingproteins
proteins provided new insights into various aspects of che- other than the ribosomal elongation factors, such as visual
momechanicalcouplingandfilamentregulation.Theinvesti- transducin, proto-oncogenes and signal transducers, such as
gatorsofthesethreeproteinfamilieswerebroughttogetherat RasandRho,arecoveredinvolumes7and9.
ameetingsponsoredbytheBiophysicalSocietyandorganized The chapters herein contain many gems for the aspiring
by Dr. Roger Cooke (see reference5 for proceedings and per- scientistandexpert,indicatingtechniquesthathavebeenused
spicacious discussions, adroitly transcribed by the attending todiscoverhowtheseproteinsoperate,whatisknownabout
students),andsincethenresearchonthesebiologicalmotors their mechanisms, what are the research frontiers. We thank
iswellintegratedanddataandtraineesaresharedsmoothly. the authors of the chapters herein, the members of our la-
Molecular motors have been a fertile proving ground for boratories and our funding sources NIH and NSF and NIST,
manybiophysicaltechniques,includingtransientbiochemical MuscularDystrophyAssociation,AmericanHeartAssociation
kineticstoelucidatereactionpathwaysandenergetics,muscle for support of molecular motor research toward the under-
fiber physiology that revealed molecular details from macro- standingthebiophysicsoftheseremarkableproteins.
scopicmechanicalmeasurements,structuralbiology,including
cryo-electronmicroscopyand,later,X-raycrystallography,and
lastly, many single molecule biophysics techniques including
References
opticaltrapsandsinglemoleculefluorescence.
Twocircumstanceshavenowfusedtheclassicalmolecular
[1] Berg,J.S.;Powell,B.C.;Cheney,R.E.Amillennialmyosincensus.Mol.Biol.
motor fields with studies on nucleic processing enzymes.
Cell2001,12(4),780–794.
Single molecule techniques that were developed for both of [2] Hirokawa,N.;Noda,Y.;Tanaka,Y.;Niwa,S.Kinesinsuperfamilymotorproteins
these areas facilitated each others’ research and meetings or- andintracellulartransport.Nat.Rev.Mol.CellBiol.2009,10(10),682–696.
ganized by Dr. Steven Block in Aspen, CO, and a Gordon [3] Lee,S.H.;Dominguez,R.Regulationofactincytoskeletondynamicsincells.
Mol.Cells.2010,29(4),311–325.
Research Conference series, initiated by Dr. Lori Goldner,
[4] Kardon,J.R.;Vale,R.D.Regulatorsofthecytoplasmicdyneinmotor.Nat.Rev.
brought the single molecule biophysicists together. Myosin, Mol.CellBiol.2009,10(12),854–865.
kinesinandmanyoftheotherNTPasessuchasGTP-binding [5] Cooke,R.Molecularmotors:Structure,mechanicsandenergytransduction.
signaling proteins, ATP synthase, helicases and viral portal Biophys.J.1995,68(4),1s–385s.
[6] Smith,C.A.;Rayment,I.Activesitecomparisonshighlightstructural
motors,haveverylowaminoacidsequencehomology,except
similaritiesbetweenmyosinandotherP-loopproteins.Biophys.J.1996,
at very short segments near their active nucleotide binding
70(4),1590–1602.
sites.HowevertheirX-raystructureshaveshownthemtohave [7] Vale,R.D.Switcheslatches,andamplifiers:commonthemesofGproteinsand
surprisingly similar secondary and tertiary structural folds in molecularmotors.J.CellBiol.1996,135(2),291–302.
their core ATP or GTP binding active sites. We refer to all of [8] Snider,J.;Houry,W.A.AAAþ proteins:diversityinfunction,similarityin
structure.Biochem.Soc.Trans.2008,36(Pt1),72–77.
themasP-loopproteinsafteraconservedsegment,alsocalled
[9] Leipe,D.D.;Wolf,Y.I.;Koonin,E.V.;Aravind,L.Classificationand
theWalkerAloop,thatencirclesandbindstothenucleotide evolutionofP-loopGTPasesandrelatedATPases.J.Mol.Biol.2002,317(1),
phosphate groups. Similarities in structure, their sequences 41–72.
ComprehensiveBiophysics,Volume4 doi:10.1016/B978-0-12-374920-8.00401-X 1
6.1 Channel Proteins – An Introduction
MMontal, University of CaliforniaSan Diego, La Jolla, CA,USA
r2012ElsevierB.V.Allrightsreserved.
References 3
Abbreviations
TM transmembrane
Glossary Lipidbilayer Thefundamentalstructuralelementofcell
Channelgating Channelproteinconformationalchanges membranesassembledbythehydrophobicappositionof
fromclosedtoopeninresponsetoanexternalforce. twolipidmonolayers.
Channelopathies Diseasesthatareassociatedwith Modulardesign Theoccurrenceofminimumunitsof
dysfunctionalchannels. structurewithspecificfunctionalattributes.
Ionchannels Ionchannels,aspecialclassofmembrane
proteinsthatallowtheselectiveandregulateddiffusionof
ionsacrossmembranes.
Ionchannels,aspecialclassofmembraneproteinsthatallow structure, and the mechanisms underlying ionic conduction,
the selective and regulated diffusion of ions across mem- selectivity,andgating.
branes,arefundamentalforcellfunctionandregulation.Their Forvoltage-gatedKþ channels(Kv)–themostextensively
design is a marvel of protein chemistry and evolution, and studiedprototype–thereisnowcompellingevidenceforthe
theirdysfunctionisattherootofdevastatinghumandiseases. occurrence of two distinct, tandemly arranged functional
Membrane channels are oligomeric proteins organized as modules,avoltagesensorandapore.2–6Theresultsofdecades
symmetricorpseudosymmetricarraysaroundacentralaque- ofexceptionalworkonthefunctionalcharacterizationofeu-
ouspore.Theultimatefunctionoftheproteinistoallowthe karyotic voltage-gated channel proteins are now com-
selectiveandregulateddiffusionofionsacrossthemembrane plemented bystructures of three voltage sensors, one from a
lipid bilayer. Membrane channels are dynamic structural de- bacterial source (KvAP) and the other two from mammalian
vices that provide a transient interface between the hydro- Kvs (Kv1.2 and a ‘paddle-chimera channel’ containing the
phobic interior of the membrane lipid bilayer and the two sensorofKv2.1inthecontextofKv1.27),andthestructureof
boundary aqueous solutions. As interfacial structures they the Kþ channel KcsA from Streptomyces lividans, which dis-
provide a solution to the thermodynamic problem of howa plays a canonical architecture of a pore module.8 How the
hydrated ion is transferred from its water shell (dielectric exquisite property of voltage sensing emerges from the
constant of 80) into and across the membrane hydrophobic underlying modular design of voltage-gated channels is still
interior thereby bypassing the low dielectric (dielectric con- unknown. Unfortunately and understandably, these crystal
stantof2)membranebarrier.Channelsareenergydissipative structures constitute only isolated frames of a dynamic spa-
structuresastheyprovideaconduitforiondiffusiondownan tiotemporal hierarchy – a movie that aims to show the fun-
electrochemical potential with rates comparable to ionic dif- damentalmechanisticprinciplesunderlyingvoltagesensingin
fusion in water. This determines that for their crucial role in ionchannels;framesforwhichwehavelittleevidenceofwhen
biologicalsystems,channelproteinsmustbetightlyregulated theyappearinthemovie.Thisaddsthedimensionoftime;it
structures.Theseconsiderations1accountforthetwohallmark is not surprising, therefore, that many have questioned whe-
properties of membrane channels, namely, selectivity and thertheframesbelongtothesamemovie.9–11Theargumentis
gating, the control of the probability of the channel being ofcrucialimportancebecausegiventhedifferentoriginofthe
openorclosed. actors,sensor,andpore,thequestionhintsattheuniversality
Awealth of biophysical and biochemical knowledge con- of the principle in all living species. The key unanswered
verged during the past three decades to establish that struc- questions are now centered on an understanding of the
turalinformationatatomicresolutionwouldberequiredfora couplingbetweenthesensorandporemodules,fundamental
rigorous understanding of how channels achieve the com- tothisdynamicviewofmodulardesign.
bination of exquisite selectivity and high throughput on the ThechapterbyShigetoshiOikidescribesinmoredetailthe
one hand, and how channels change conformation from functional aspects that are characteristic of voltage-gated
closed to open in response to an external force (transmem- channels, focusing on Kþ channels. Mark Sansom offers a
brane potential or ligand binding) on the other. Major ad- perspective to an understanding of Kv function by means of
vanceshavebeenachievedwithrespecttomembraneprotein computational studies of both structure and dynamics. The
ComprehensiveBiophysics,Volume6 doi:10.1016/B978-0-12-374920-8.00601-9 1
2 ChannelProteins – AnIntroduction
chapter by Yasushi Okamura reviews evidence for the occur- rival bacteria to human cells. The molecular architecture of
renceofavoltagesensorasanindependentlyfoldedmodule thesenanomachinesunderpinningtheirnoxiousfunctionisa
and its operation as a voltage-gated, proton-selective, and triumph of protein design. Examples of these sophisticated
Zn2þ-sensitive channel.12–16 Remarkably, such a sensor channelsformedbybacterialtoxinsareillustrated byRoland
module is present in other membrane protein families ex- Benz.
cludingionchannels,aselegantlyshownintheC.intestinalis Virus-encoded ion channels emerge as a family of prim-
voltage-sensor-containing phosphoinositide phosphatase, ordialproteinsthatpredatedthehighlyselectiveandregulated
therebyconferringvoltagesensitivityontheenzyme.17 channel proteins of Bacteria and Eukarya. Phylogenetic an-
The collective knowledge validates the modular design of alysis indicates that the chlorella Kcv channel is a very
channel proteins.1,2 The provocative prospect of establishing primitive Kþ channel and may represent the ancestor of the
the occurrence of minimum units of structure with specific archetypal KcsA channel,8 namely, the ancestor of the pore
functionalattributesopensatestablepathtoassesstheorigin module.TheseaspectsarereviewedbyGerhardThiel.Froma
of channel diversity emerging from module shuffling and protein evolution viewpoint, the Kcv channel is therefore
optimizationbymutationevents,andtoanunderstandingof morecomplexthantheM2proteinofinfluenzaAvirusorVpu
thesurfacefeaturesunderlyingthecompatibilitybetweenthe of HIV-1 virus. These two channel proteins may represent a
two modules responsible for the functional coupling of the limit of simplicity: a single transmembrane (TM) helix per
sensortotheporeand,ultimately,ofchannelgating. polypeptidewithaninherentpropensitytooligomerize.18The
Thisfundamentalproteindesignisconservedfrombacteria beauty of this essential design resides in the fact that the
to plants; it is ubiquitous, as it extends from plasma mem- channelactivityisathermodynamicconsequence ofthenat-
branestointracellularmembranes;andofdiverseselectivityas ural tendency of the membrane-embedded, amphipathic TM
it covers monovalent and divalent cations. These distinctive helixtoaggregateinordertooptimizeitsinteractionswithin
aspectsareanalyzedbyNobuyukiUozumi,PatrickHogan,and the hydrophobic interior of the bilayer, thereby generating a
Baruch Minke. Tsun-Yu Cheng and Tzyh-Chang Hwang’s helical bundle, that is, the structural blueprint for the chan-
chapter highlights the major differences in design between nel.19Thisisfascinatinginsofarasitallowsonetoconjecture
cationchannelsandCl(cid:2)channelsaswellastheemergingview iffunctionaldiversitywasinitiallyacquiredbyevolvingfroma
thataclassofCl(cid:2)channelsmayoperateasantiportersinthe singleTMmotif,asinVpu(non-selectivechannel),tothetwo
context of vesicular membranes, akin to their bacterial TMs with a linker motif, as in Kcv and KcsA (Kþ-selective
homologues. And Mark Yeager outlines the application of channel), and much later into fusion with a voltage sensor
high-resolution,single-particleelectronmicroscopytodecode moduletogeneratethesixTMmotifsfoundinvoltage-gated
themoleculararchitectureofconnexinchannels. channels of Prokarya and Eukarya(voltage-gated and cation-
Channelgatingisnotoriouslyassociatedwithvoltage-gated selectivechannels).
channelsofexcitablecells.Ligand-gatedchannelsconstitutea Marco Colombini introduces a novel class of membrane
secondprominentclassofionchannelsthatareregulatedby channels,formednotbyproteinsbutbyceramides.Theseare
bindingofaligand.Amongthesearetheionotropicglutamate pore-forming structures self-assembled from hundreds of
receptors, which mediate excitatory synaptic transmission in monomers that form dynamic aqueous pores with dimen-
the central nervous system. Steve Traynellis et al. present a sions that exceed the thickness of a lipid bilayer. Ceramide
criticaloverviewofthestructuralfeaturesthatendowthisclass channels are in turn regulated by auxiliary proteins and dis-
of channels with their functional attributes that make them play allosteric-like behavior, including positive and negative
uniquely suited to operate in neurotransmission. Channel cooperativity.
gating is also exhibited by prokaryotic proteins. Channel Seminal contributions to the development of our know-
proteinsofprokaryotesevolvedbeforethoseofEukarya,and ledge of membrane protein structure have deepened our
theiramenabilitytoexperimentalanalysishasyieldedawealth understanding of how the structure of membrane channels
of insightful information about structure, dynamics, and determines their function. It is fair to predict that this infor-
regulation of channel activity. A salient example is that of mationwillhavebiomedicalrelevanceandwillbringuscloser
mechanosensitive channels, a class of channels for which toa detailedunderstandingofhowstructuralalterationspro-
gatingisregulatedbythedirectsensingofmembranetension duce disease states. Numerous diseases are associated with
within the lipid bilayer. This fascinating class of channels is dysfunctional channels. The term channelopathies has been
reviewedbyBorisMartinac. coined to underscore this diverse group of diseases, which
Regulationofchannelactivityisalsoaccomplishedbythe encompassepilepsy,arrhythmia,myotonia,periodicparalysis,
attachment of diverse modules to the basic design. Cyclic cysticfibrosis,anddeafness,amongothers.20Drugdesignand
nucleotide binding domains are a casein point. Inaddition, discovery aimed at compensating such defects may be facili-
membrane channels are involved in protein-protein inter- tatedbytheavailabilityofhigh-resolutionstructuresofmem-
actions localized at the membrane interface, most promin- branechannels.Drugdiscoverywithionchannelsasthetargets
entlyontheintracellularmilieu.Assuch,largemulti-protein isnowafertilefieldofscientificendeavor.Theexpectationthat
complexes are assembled that express different functional wellinexcessof1000newpotentialdrugsjustinthefieldof
consequences.Amongthemoststudiedregulatorsareprotein neuroscience will be derived from genomics and that more
kinasesandphosphatases,Gproteins,andGprotein-coupled than90%ofthesewillbetargetedtochannelsandreceptors,
receptors. willrequirestructuralknowledgeofthedrugbindingpocket.
Pore-forming proteins are ubiquitously produced by bac- In the light of the pace of progress and with the con-
teria and used for intoxication of target cells, ranging from vergenceofconceptualandtechnicalbreakthroughs,wenow
ChannelProteins – AnIntroduction 3
stand at the threshold of an exciting phase in the under- [8] Doyle,D.A.;MoraisCabral,J.;Pfuetzner,R.A.;Kuo,A.;Gulbis,J.M.;
standing of the function of membrane channels in terms of Cohen,S.L.;Chait,B.T.;MacKinnon,R.Thestructureofthepotassium
channel:molecularbasisofKþ conductionandselectivity.Science1998,
theirstructure.Thenewknowledgehasgivenfurthersupport
280,69–77.
to nature’s conservation of the pillar principles of protein
[9] Bezanilla,F.Ionchannels:fromconductancetostructure.Neuron2008,60,
scienceinthecontextoftheuniqueenvironmentconferredby 456–468.
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6.2 Structure-Function Correlates of Glutamate-Gated Ion Channels
KBHansen, Emory University Schoolof Medicine,Atlanta, GA, USA
LP Wollmuth,Stony Brook University, StonyBrook,NY, USA
SFTraynelis, Emory University SchoolofMedicine, Atlanta, GA,USA
r2012ElsevierB.V.Allrightsreserved.
6.2.1 Structureof Glutamate-GatedIon Channels 4
6.2.1.1 The GlutamateReceptor Complex 5
6.2.1.2 SubunitStoichiometry 6
6.2.1.3 LigandBinding Domain 7
6.2.1.4 Amino-Terminal Domain 9
6.2.1.5 Pore-Forming Domain 11
6.2.1.6 Carboxy-Terminal Domain 12
6.2.1.7 Auxiliary Subunits 12
6.2.2 GlutamateReceptorActivation andModulation 12
6.2.2.1 Activation andDeactivation 12
6.2.2.2 Mechanisms Linking AgonistBindingtoGating 13
6.2.2.3 Molecular Mechanism ofPartial Agonism 13
6.2.2.4 Molecular Mechanism ofDesensitization 15
6.2.2.5 Allosteric Modulatorsatthe LigandBindingDomain Dimer Interface 17
6.2.2.6 IonsActing attheLigandBindingDomain Dimer Interface 18
6.2.2.7 Divalent IonsActing as Negative Allosteric Modulators 18
6.2.2.8 ProtonsActing as NegativeAllosteric Modulators 19
6.2.3 GlutamateReceptorGating 19
6.2.3.1 Structural Determinants oftheGate 19
6.2.3.2 Molecular Determinants ofGating 20
6.2.3.3 Molecular Determinants Linking AgonistBindingto Gating 20
6.2.4 GlutamateReceptorPermeation 21
6.2.4.1 Structural Features oftheIonPermeation Path 21
6.2.4.2 Mechanisms ofIonSelectivity 23
6.2.4.3 Mechanisms ofVoltage-Dependent ChannelBlock 23
Acknowledgments 24
References 24
Abbreviations NT Nterminus
ATD amino-terminaldomain TARP transmembraneAMPAreceptorregulatory
LBD ligandbindingdomain protein
LTP long-termpotentiation TMD transmembranedomain
6.2.1 Structure of Glutamate-Gated Ion Channels composition of postsynaptic glutamate receptors and vari-
ations in their interaction partners allow cells to alter their
Withinthemammalianbrain,spinalcord,andretina,excita- synaptic response kinetics to suit the role played by each
torysynaptictransmissionismediatedbyglutamatereceptors, synapseinthenetwork.
whichareligand-gatedionchannels.Becauseoftheiressential Glutamate receptor subunits are classified into at least
role in mediating the excitatory postsynaptic response, these threeclearcategoriesdefinedbypharmacologyandstructure:
receptorsparticipateinvirtuallyallaspectsofbrainfunctionas AMPAreceptors,kainatereceptors,andNMDAreceptors(see
well as multiple neurological diseases. Following release of Collingridge et al.1 for nomenclature). In addition, the delta
vesicularglutamateintothesynapticcleft,glutamatebindsto subunitsformafourthclassofglutamatereceptors,although
the postsynaptic glutamate receptors and triggers a rapidly their role in brain function remains unclear. AMPA receptors
risingincreaseinconductancethattypicallydecaysrapidlydue showrapidresponsekineticsthatlimitthesynapticconduct-
to deactivation of the receptor complex and dissociation of ance waveform to a duration typically of only a few milli-
glutamate. The time course and response amplitude of the seconds. The resulting excitatory postsynaptic potential rises
excitatory synaptic current reflect the biophysical properties and decays rapidly.2 Kainate receptors can play important
and density of the receptors as well as the time course of rolesatbothpre-andpostsynapticsitesinthecentralnervous
glutamate release and uptake. The varying subunit system. Postsynaptic kainate receptors mediate an excitatory
4 ComprehensiveBiophysics,Volume6 doi:10.1016/B978-0-12-374920-8.00611-1
Structure-Function Correlatesof Glutamate-GatedIon Channels 5
postsynaptic current with a slower time course than that for These data are consistent with the idea that the glutamate
AMPA receptors, and a comparable and generally faster time receptor ligand binding domain is a dimer-of-dimers. The
course than that for NMDAreceptors,depending on subunit twofold rotational symmetry differs from that observed in
composition.3 NMDA receptors are co-localized with AMPA tetrameric Kþ channels and pentameric nicotinic acetyl-
receptors at virtually all central synapses,4 and they typically choline receptors, for which the subunit arrangement neces-
mediate a slower component of the synaptic current. NMDA sitates a close relationship between subunit number and
receptors are normally blocked in a voltage-dependent man- rotationalsymmetry.18–21
ner by extracellular Mg2þ, enabling coincident detection of AstructureofatetramericAMPAreceptor(GluA2)at3.6A˚
bothdepolarization(whichrelievesMg2þ block)andthere- resolution verified the twofold symmetry, showing that the
leaseofglutamate.NMDAreceptorshavelongbeenknownto extracellularN-terminaldomainsandligandbindingdomains
trigger changes in synaptic strength. When postsynaptic areorganizedasdimers-of-dimers.Thechannelpore-forming
NMDA receptors are activated during high-frequency stimu- domainofhomomericGluA2hasfourfoldsymmetry,similar
lation, they mediate an increase of intracellular Ca2þ within to potassium channels.12 The symmetry mismatch between
the dendritic spine, which initiates long-lastings changes in the extracellular domain and the transmembrane domain
postsynaptic strength that is often referred to as long-term arises because the receptor contains two conformationally
potentiation (LTP). NMDA receptor-dependent LTP has been distinct subunits, denoted A/C and B/D subunits (Figure 2).
the most widely studied form of synaptic plasticity, and has The different conformations of identical subunits may result
been suggested to be the cellular basis of learning and in a different coupling mechanism during the process of
memory.5,6 channelgating,whichcouldhaveimportantimplicationsfor
receptor function. The most extensive interdomain contacts
fortheN-terminaldomainareformedbetweenA/BandC/D
6.2.1.1 TheGlutamateReceptorComplex
subunits. Interestingly, the key features of this contact are
Glutamatereceptorsaremembrane-spanningproteinsthatare similartothoseobservedinthecrystallographicstructuresof
multimeric complexes of four subunits that form a cation- isolated N-terminal domains for the GluA2 and GluK2 ATD
selective pore through the plasma membrane. All glutamate dimer.22–24AsecondcontactregionisformedbetweenBand
receptor subunits share considerable sequence identity and DsubunitsoftheA/BandC/Ddimers(Figure2).Theligand
presumably a similar molecular architecture (Table 1). Glu- bindingdomainsarearrangedasA/DandB/Cdimers,withA
tamate receptor subunits are composed of four semi- andCsubunitcontact.Thus,theunexpectedsubunitcrossover
autonomous domains, including an extracellular amino- creates a unique arrangement at the levels of both the
terminal domain, an extracellular ligand binding domain, a N-terminalandtheligandbindingdomains.
transmembrane pore-forming domain, and an intracellular Theglutamatereceptorporesharesweakhomologytothe
carboxy-terminal domain (Figure 1). Most domains exhibit tetrameric Kþ channels,andthus it waspredicted to possess
modestsequencehomologytobacterialproteinswithknown similar structural arrangement before crystallographic data
structures and somewhat related function.7–11 Crystallo- verified this prediction.7,10,11 The glutamate receptor trans-
graphic coordinates are available for a membrane-spanning membrane domain consists of three membrane-spanning
AMPA receptor,12 in addition to isolated extracellular helices (M1, M3, and M4) and a re-entrant pore-lining loop
N-terminalandligandbindingdomains. (M2).12 Glutamate receptors M1–M3 assemble to form a
Single particle images of AMPA receptors obtained by structure that is similar to aninvertedKþ channel pore. The
electronmicroscopyshowthereceptorsat20–40A˚ resolution, M4transmembranehelix,whichdoesnothaveacounterpart
revealing a twofold rotational symmetry in some cases.13–17 inpotassiumchannels,primarilymakescontactswiththeion
Table1 Sequenceidentityofresiduesinglutamatereceptorsubunitsa
Receptorsubunits ATD(%) LBD(%) TMD(%) CTD(%) All(%)
GluA1–4 35(89) 80(100) 87(98) 9(60) 54(90)
GluK1–5 16(67) 53(92) 56(96) 0.0(13) 29(70)
GluN1
GluN2A–D 1(24) 19(68) 14(81) 0.0(2.9) 5(29)
GluN3A–B
GluN2A–D 19(76) 63(96) 73(99) 2(47) 25(70)
GluD1–2 60(60) 62(62) 54(54) 34(34) 54(54)
Allsubunits 0.2(15) 6(48) 10(55) 0.0(0.2) 2(19)
aNumbersarethepercentagesofresiduesintheregionsthatareidenticalinallsubunitswithinthegroup.Numbersinparenthesesarethepercentagesofresiduesthatareidentical
in50%ofthesubunitsinthegroup(i.e.,conserved).InGluA2,theregionsweredefinedasaminoacids1–397(signalpeptideandATD),415–527(S1),535–647(M1M2M3),
653–794(S2),810–838(M4),and839–884(CTD)usingthestructuresoftheisolatedGluA2LBD80andthemembrane-spanningtetramericGluA212asguides.
ATD,amino-terminaldomain;CTD,carboxy-terminaldomain;LBD,ligandbindingdomain,composedofS1andS2regions;TMD,transmembranedomain,composedofM1M2M3
andM4.
6 Structure-FunctionCorrelates of Glutamate-GatedIon Channels
ATD
1 1
7 4
0 0
90° Å Å
90°
LBD
Out
TMD
In
50 Å 110 Å
Figure1 Theglutamatereceptorcomplex.Structureofthemembrane-spanningtetramericGluA2AMPAreceptor(3.6A˚;PDBcode3KG2).
Glutamatereceptorsubunitsarecomposedofextracellulardomainsreferredtoastheamino-terminaldomain(ATD)andtheligandbinding
domain(LBD),thetransmembranepore-formingdomain(TMD),andanintracellularcarboxy-terminaldomain(notincludedinthestructure).
channel core (M1–M3) of an adjacent subunit. The obser- AMPA and most kainate receptor subunits can form homo-
vation that identical subunits within the homomeric AMPA andheteromericassemblies,althoughGluK4andGluK5only
receptor structure adopt distinct conformations within the function when co-expressed with GluK1 to GluK3.43,44 The
multimeric complex has not previously been reported or delta receptors GluD1 and GluD2 form homomeric as-
predicted for ligand-gated ion channels.12 The subunit cross- sembliesthatcannotbeactivatedbyanyknownligands.45–48
overbetweentheextracellulardomainsismediatedmostlyby NMDA receptor activation requires simultaneous binding
the linker regions between the N-terminal domain and the of the co-agonists glutamate and glycine (reviewed by Tray-
ligandbindingdomain.TheselinkerswithintheA/Csubunits nelisetal.49).TheGluN1andGluN3subunitscontainglycine
adoptamorecompactconformationrelativetothelinkersof binding sites,50–52 whereas the GluN2 subunits contain the
the B/D subunits, which are extended. Previous studies have glutamate binding site.51 NMDA receptors comprise two
suggested that this segment controls open probability of GluN1subunitstogetherwithtwoGluN2subunits;53insome
NMDA receptors.25,26 Interestingly, the symmetry mismatch cases,aGluN3subunitisthoughttoco-assemblewithGluN1
between the ligand binding domain and the pore-forming andGluN2.54,55NMDAreceptorscanalsobeformedwithtwo
domainismediatedprimarilybythelinkers,whichadopttwo distinct GluN2subunits,whichare often referredto as trihe-
different conformations corresponding to the A/C subunits teromeric receptors. Although theoretically any combination
andtheB/Dsubunits.Avarietyofstudieshaveaddressedthe of GluN2 subunits should be able to co-assemble into trihe-
functional significance of the linkers between the pore-form- teromeric receptors, available data to date support the as-
ingelementsandtheligandbindingdomains.27–34Although sembly of native GluN1/GluN2A/GluN2B, GluN1/GluN2A/
we assume key elements of the AMPA receptor structure will GluN2C, GluN1/GluN2B/GluN2D, and GluN1/GluN2A/
transfertobothkainateandNMDAreceptors,thisassumption GluN2Dreceptorsindifferentbrainregions.56–70Despitethe
hasyettobetested. physiological prevalence of triheteromeric receptors, only
limiteddataareavailableontheirbiophysicalproperties.71–73
The GluN3 subunits do not form functional homomeric
receptors.74 Although recombinant GluN1/GluN3 receptors
6.2.1.2 SubunitStoichiometry
form glycine-activated cation-permeable receptors in some
The glutamate receptors, like potassium channels, are tetra- heterologous expression systems,74 native neuronal glycine-
mers.12,14,35–38 Without exception, functional channels are activatedcation-permeablereceptorshavenotbeendescribed.
assemblies of subunits within the same receptor class.39–43 Co-expression of GluN3 with GluN1/GluN2 in Xenopus oo-
Glutamatereceptorsubunitscanbesubdividedintothesame cytes reduces NMDA- and glutamate-activated current ampli-
four classes mentioned previously on the basis of agonist tudes, suggesting that either triheteromeric GluN1/GluN2/
pharmacology and sequence similarity: AMPA receptors GluN3 receptors have a reduced conductance or GluN3 hin-
(GluA1 to GluA4), kainate receptors (GluK1 to GluK5), ders trafficking or assembly.54,55,75–78 Native triheteromeric
NMDAreceptors(GluN1,GluN2AtoGluN2D,GluN3A,and GluN1/GluN2/GluN3 receptors have been suggested to exist
GluN3B), and delta receptors (GluD1 and GluD2). Both basedontheobservationofneuronalsingle-channelcurrents