MEMBRANE
PROTEINS
Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso
Index
1.
Introduction
•
What is cell membrane?
•
Integral Proteins
•
Crystallization
2.
Transmembrane Proteins
•
β - Barrels
•
α-Helical
3.
GPCRs
•
What are G protein – coupled receptors?
•
Classification
•
Sequence conservation
•
Class A
•
SCOP
•
Chronology
•
Alignments
•
β2-Adrenergic Receptor
•
Extracellular Loop 2 (ECL2)
•
Ligand binding site
•
Ionic Lock
•
Activation
4.
5.
Conclusions
Bibliography
1. Introduction
What is cell membrane ?
 Composed of a phospholipid bilayer with a collage of many different proteins, lipids and
carbohydrates
 It has many functions
Membrane Proteins:
 They can be classified into two main categories: peripheral and integral proteins
Fig2. Cell membrane with integral proteins embedded.
Fig1. Cell membrane with peripheral proteins.
1. Introduction
Transmembrane proteins
Extracellular region
Transmembrane region
Intracellular region
Fig3. Cell membrane with a transmembrane protein.
Domain
Functions
Extracellular
Involved in cell – cell signaling
Let interactions with other molecules
Transmembrane
Forms channels and pores
Intracellular
Activates intracellular signaling pathways
1. Introduction
CRYSTALLIZATION. Why is it so complicated?
 Difficulties in handling and crystallization of integral membrane proteins reside in
the amphiphatic nature of their surface
They have hydrophobic transmembrane regions and two hydrophillic regions,
one of each side of the membrane
As a result, membrane proteins are not soluble in aqueous buffers or in organic
solvents
Membrane protein structural biology:
The most challenging targets in structural biology
1. Introduction
CRYSTALLIZATION. Why is it so complicated?
Trying to solubilize and crystalize transmembrane proteins:
To solubilize transmembrane proteins is necessary to add amphipathic
molecules such as detergents to cover the transmembrane region
Addition of detergents
doesn’t guarantee stably
solubilized membrane
proteins
Nowadays, crystallization techniques try to
add lipids to the detergents to improve the
solubilization process
Index
1.
Introduction
•
What is cell membrane?
•
Integral Proteins
•
Crystallization
2.
Transmembrane Proteins
•
β - Barrels
•
α-Helical
3.
GPCRs
•
What are G protein – coupled receptors?
•
Classification
•
Sequence conservation
•
Class A
•
SCOP
•
Chronology
•
Alignments
•
β2-Adrenergic Receptor
•
Extracellular Loop 2 (ECL2)
•
Ligand binding site
•
Ionic Lock
•
Activation
4.
5.
Conclusions
Bibliography
2. Types of transmembrane proteins
β - Barrels
• Are found in the outer membranes of Gram – negative bacteria
• It is formed by an array of beta – strands arranged in an antiparallel manner
Extracellular
Extracellular
Intracellular
Fig4. NalP protein
Fig6.
Fig5. OmpA protein
Fig7.
2. Types of transmembrane proteins
α - Helical
Extracellular
Intracellular
Fig8. PufX protein
Bitopic
Polytopic
Fig9. Rodhopsin
• It contains a hydrophilic
domain on each side of the
membrane
• It contains a multiple
hydrophilic domains on both
sides of the membrane
Fig10. Bitopic proteins and Polytopic proteins
Index
1.
Introduction
•
What is cell membrane?
•
Integral Proteins
•
Crystallization
2.
Transmembrane Proteins
•
β - Barrels
•
α-Helical
3.
GPCRs
•
What are G protein – coupled receptors?
•
Classification
•
Sequence conservation
•
Class A
•
SCOP
•
Chronology
•
Alignments
•
β2-Adrenergic Receptor
•
Extracellular Loop 2 (ECL2)
•
Ligand binding site
•
Ionic Lock
•
Activation
4.
5.
Conclusions
Bibliography
3. GPCRs
What are G protein-coupled receptors ?
Integral membrane proteins are characterized by seven α-helix transmembrane
domains with an extracellular N terminus and a cytoplasmastic C terminus
Mediate the effect
of numerous ligands
Activate different
signaling paths
The most important family
of membrane proteins
One of the most important
types of receptors
GPCR
3. GPCRs
Classification
Fig12. Scheme of structure of the class B
Fig11. Scheme of structure of the class A
Fig13. Scheme of structure of the class C
3. GPCRs
Sequence Conservation
A_adenosine
A_beta-1
A_beta
A_rhodopsin
B_Glucagon
B_Pituitary
B_Parathyroid
B_Vasoactive
C_Gamma-aminob
C_Metabotropic
C_Metabotropic
C_Taste
P..FAI..TI.........---.........STGFCA..ACHGCLF.IACFVLVLTQSSIFSLLA..IA..IDRY..IA.....I--RIPL-RY-NGLVT
P..FGA..TI.........VVW.........GRW-EY..GSFFCEL.WTSVDVLCVTASIETLCV..IA..LDRY..LA.....I--TSPF-RY-QSLLT
P..FGA..AH.........ILM.........KMW-TF..GNFWCEF.WTSIDVLCVTASIETLCV..IA..VDRY..FA.....I--TSPF-KY-QSLLT
T..STL..YT.........SLH.........GYF-VF..GPTGCNL.EGFFATLGGEIALWSLVV..LA..IERY..VV.....VCKPMSN---F--RFG
R..YSQ..KIg......ddLSV.........STW-LSdgAVAGCRV.AAVFMQYGIVANYCWLLVegLY..LHNL..LG.....L-ATLPERSFF----A..ISV..FI.........---.........KDWILY..AEQDSN-.HCFISTVECKAVMVFFHY..CV..VSNY..FW.....L-FIEGL--YLFTLLV
Y..SGA..TLdeaerlteeELRaiaqappppATA-AA..GYAGCRVaVTFFLYFLATNYYWILVE..--..-GLY..LH.....SLIFMAF---FS---E
IkdLAL..FD.........SGE.........SDQCSE..GSVGCKA.AMVFFQYCVMANFFWLLV..EG..LYLYtlLA.....V-SFFSERKYF----P..LGLdgYH.........IGR.........NQF---..-PFVCQ-.-ARLWLLGLGFSLG----..--..---Y..GS.....MFTKIWW---VHTVFT
P..FTL..IA.........--K.........PTT---..--TSCYL.QRLLVGLSSAMCYSALVT..KTnrIARI..LAgskkkICTRKP-------RFM
P..STA..VC.........TLR.........RLG---..--LGTAF.SVCYSALLTKTN------..-R..IARI..FG.....-GAREGA---QRPRFI
P..TRP..AC.........LLR.........QALFAL..G-----F.TIFLSCLTVRS--FQLII..I-..----..FK.....FSTKVPTF--YHAWVQ
A_adenosine
A_beta-1
A_beta
A_rhodopsin
B_Glucagon
B_Pituitary
B_Parathyroid
B_Vasoactive
C_Gamma-aminob
C_Metabotropic
C_Metabotropic
C_Taste
GTRAKGIIAIC...WVL.SFAIG-.LTPM....LGW-N----NCGQPKEGKNHSQGCGEGQVAC.LF-EDV--.-----VPMNYMVYFN..FFA.CVLVP
RARARGLVCTV...WAI.SALVSF.LPIL....MHWWRAESDEA--RR--CYNDPKC------C.D-F--VT-.------NRAY-AIAS..SVV.SFYVP
KNKARVIILMV...WIV.SGLTSF.LPIQ....MHWYRATHQEA--IN--CYANETC------C.D-F--FT-.------NQAY-AIAS..SIV.SFYVP
ENHAIMGVAFT...WVM.ALACAA.-PPL....AGWS-------------R---YIPEGLQCSC.GID--YYT.LKPEVNNESF-VIYM..FVV.HFTIP
-----------...---.SLYLG-.----....IGW------GAPMLFVVPWAVVK-------C.LF-ENV-Q.CWTSNDNMGFWWILR..FPV.FLAIETFFPERRYFY...WYT.IIGWGT.PTVC....VTVW------A-TLR--LYFDDTG------CwDM-NDST-.------ALWW-VIKG..PVVgSIMVN
KKYLWGFTVFG...WGLpAVFVAV.----....--WV---SVRA------TLANTG-------C.W-D--LS-.----SGNKKW-IIQV..PIL.ASIV----WGYILIG...WGV.PSTFT-.----....MVWT-----IA-RIH---FEDYG-------C.W-D---T-.-----INSSLWWIIKgpILT.SILVN
KKEEKKEWRKTlepWKL.YATVGL.LVGMdvltLAIWQIVDPLHRTIE--TFAKEEP-----KE.DID--VSIlPQLEHCSSRKMNTWL..GIF.YGYKG
SAWAQVIIA--...---.SILISVqLTLV....VTLI---------IM--E--PPMP-------.-IL--SY-.---PSIKEVYLICNT..SNL.GVVAP
SPASQVAIC--...--L.ALISGQ.LLIV....VAWL---VVEA----PGTGKETAP-------.ER-REVV-.----TLRCNH-RDAS..MLG.SLAYN
NHGAGLFVMIS...SAA.QLLIC-.LTWL....VVW---TPLPA---R--EYQR-FP------H.LVMLECT-.---ETNSLGF-----..-IL.AFLYN
Fig14. Fragment of sequence alignment of the three main classes of GPCRs
3. GPCRs
Class A
Fig16. Scheme of structure of the class B
Fig15. Scheme of structure of the class A
Fig17. Scheme of structure of the class C
3. GPCRs
Class A. SCOP
Fig 18. Class A classification from SCOP (Structural Classification of Proteins)
3. GPCRs
Class A. Chronology
Before 2000
2000
2007
Rhodopsin
Palczewski et al. Aug. 2000
2008
2008-2012
β1 -Adrenergic Receptor
Warne et al. July 2008
2-Adenosine Receptor
Jaakola et al. Oct. 2008
β2-Adrenergic Receptor
Cherezov et al. Nov. 2007
D1-R
H1-R
SPS1
…
3. GPCRs
adenosine
AVLAILGNVLVCWAVWL..NSNLQNV...TNYFVVSLAAADIAVGVLAIP.FAITI---STGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAI--RIPL-RY-NGLV.TGTR.A.KGIIAICWVLSFAIG-LTPMLGW-N---beta-1
VLLIVAGNVLVIVAIAK..TPRLQTL...TNLFIMSLASADLVMGLLVVP.FGATIVVWGRW-EYGSFFCELWTSVDVLCVTASIETLCVIALDRYLAI--TSPF-RY-QSLL.TRAR.A.RGLVCTVWAISALVSFLPILMHWWRAESD
beta
VLAIVFGNVLVITAIAK..FERLQTV...TNYFITSLACADLVMGLAVVP.FGAAHILMKMW-TFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAI--TSPF-KY-QSLL.TKNK.A.RVIILMVWIVSGLTSFLPIQMHWYRATHQ
chemokine_r FLTGIVGNGLVILVMGY..QKKLRSM...TDKYRLHLSVADLLFVIT-LP.FWAVDAVANWY--FGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAI-VHATN-S-Q--RP.RKLL.AeKVVYVGVWIPALLLT-IPDFI--F-ANVS
D2
IAVIVFGNVLVCMAVSR..EKALQTT...TNYLIVSLAVADLLVATLVMP.WVVYLEVVGEW-KFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAV--AMPML--YNTRYsSKRR.V.TVMISIVWVLSFTISC-PLLFGL-N---galanin
FLLGTVGNGLVLAVLLQpgPSAWQEPgstTDLFILNLAVADLCFILCCVP.FQATIYTLDAW-LFGALVCKAVHLLIYLTMYASSFTLAAVSVDRYLAV--RHPL-R-SRALR.TPRN.A.RAAVGLVWLLAALFSA--PYLSYY----histamine
ILITVAGNVVVCLAVGL..NRRLRNL...TNCFIVSLAITDLLLGLLVLP.FSAIYQLSCKW-SFGKVFCNIYTSLDVMLCTASILNLFMISLDRYCAV--MDPL-RY-PVLV.TPVR.V.AISLVLIWVISITLSFLSIHLGW-NSRNE
melatonin
TAVDVVGNLLVILSVLR..NRKLRNA...GNLFLVSLALADLVVAFYPYP.LILVAIFYDGW-ALGEEHCKASAFVMGLSVIGSVFNITAIAINRYCYICHSMAYHRIYR---.-RWH.T.PLHICLIWLLTV-VALLPNFFVGSL---muscarinic
SLVTIIGNILVMVSIKV..NRHLQTV...NNYFLFSLACADLIIGVFSMNlYTLYT-VIGYW-PLGPVVCDLWLALDYVVSNASVMNLLIISFDRYFCV--TKPL-TY-PVKR.TTKM.A.GMMIAAAWVLSFILWA-PAILFW-----delta
CAVGLLGNVLVMFGIVR..YTKMKTA...TNIYIFNLALAD-ALATSTLP.FQSAKYLMETW-PFGELLCKAVLSIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALD-FR---.TPAK.A.KLINICIWVLASGVGV-PIMVMAV--TRP
kappa
FVVGLVGNSLVMFVIIR..YTKMKTA...TNIYIFNLALAD-ALVTTTMP.FQSTVYLMNSW-PFGDVLCKIVISIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALD-FR---.TPLK.A.KIINICIWLLSSSVGISAIVLGG-TKVRE
Mu-type
CVVGLFGNFLVMYVIVR..YTKMKTA...TNIYIFNLALAD-ALATSTLP.FQSVNYLMGTW-PFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALD-FR---.TPRN.A.KIINVCNWILSSAIG-LPVMFMAT----orexin
FVVALVGNTLVCLAVWR..NHHMRTV...TNYFIVNLSLADVLVTAICLP.ASLLVDITESW-LFGHALCKVIPYLQAVSVSVAVLTLSFIALDRWYAICHPLLF-K--S---.TARR.A.RGSILGIWAVSLAIMV--PQAAVM----rhodopsin
IVLGFPINFLTLYVTVQ..HKKLRTP...LNYILLNLAVADLFMVLGGFT.STLYTSLHGYF-VFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSN---F--RF.GENH.A.IMGVAFTWVMALACAA-PPLAGWS----5-hydroxytryp IFCAVLGNACVVAAIAL..ERSLQNV...ANYLIGSLAVTDLMVSVLVLP.MAALYQVLNKW-TLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAI--TDPI-DYVN-KR.TPRR.A.AALISLTWLIGFLIS-IPPMLGW-R--TP
somatostatin CAAGLGGNTLVIYVVLR..FAKMKTV...TNIYILNLAVAD-VLYMLGLP.FLATQNAASFW-PFGPVLCRLVMTLDGVNQFTSVFCLTVMSVDRYLAV--VHPL---SSARW.RRPRvA.KLASAAAWVLSLCMS-LPLLV--F-ADVQ
TM1
TM2
TM3
adenosine
NCGQP.KEGKNHSQGCGEGQVACLF-EDV-------VPMNYMVYFNF...FACVLVPLLLMLGVYLR....I...FLAAR...
beta-1
EA--R.R--CYNDPKC------CD-F--VT-------NRAY-AIASS...VVSFYVPLCIMAFVYLR....V...FREAQ...
beta
EA--I.N--CYANETC------CD-F--FT-------NQAY-AIASS...IVSFYVPLVIMAFVYSR....V...FQEAK...
chemokine_r EA--D.D--R---YI-------CDRF--YP-------NDLWVVVFQFqhiMVGLILPGIVILSCYCI....I...ISKL-...
D2
NA---.---DQNE---------CIIA-----------NPAF-VVYSS...IVSFYVPFIVTLLVYIK....I...YIVLR...
galanin
--GTV.R--YGALEL-------CV-P--AW---EDARRRAL-DVATF...AAGYLLPVAVVSLAYGRtlrfL...WAAVG...
histamine
TS---.---KGNHTTS-----KCKVQ--V--------NEVY-GLVDG...LVTFYLPLLIMCITYYR....I...FKVAR...
melatonin
-----.---EYDPRIY-----SCT-F--IQ-----TASTQY-TAAVV...VIHFLLPIAVVSFCYLR....IwvlVLQAR...
muscarinic
QF--I.VGVRTVEDGE------CYIQ--FF------SNAAV-TFGTA...IAAFYLPVIIMTVLYWH....I...SRASK...
delta
RDGAV.VCML-QFPSP-----SWYWD-TVT------------KICVF...LFAFVVPILIITVCYGL....M...LLRLR...
kappa
DVDVI.ECSL---QFP-------D-D-DYS------WWDLFMKICVF...IFAFVIPVLIIIVCYTL....M...ILRLK...
Mu-type
----T.K--YRQGSID------CTLT--FS--HPTWYWENLLKICVF...IFAFIMPVLIITVCYGL....M...ILRLK...
orexin
ECSSVlP--ELANRTR--LFSVCD--ERWA---DDLYPKIY-HSCFF...IVTYLAPLGLMAMAYFQ....I...FRKLW...
rhodopsin
-----.---R---YIPEGLQCSCGID--YYTLKPEVNNESF-VIYMF...VVHFTIPMIIIFFCYGQ....L...VFTVK...
5-hydroxytryp ED---.---R-SDPDA------CTISKDH----------GY-TIYST...FGAFYIPLLLMLVLYGR....I...FRAARfri
somatostatin EGGTC.NASW---PEP------VGLW-----------GAVF-IIYTA...VLGFFAPLLVICLCYLL....I...VVKVR...
TM5
TM4
HAA....K...SLAIIVGLFALCWLPLHI...IN....CFT...F.FC-P
KAL....K...TLGIIMGVFTLCWLPFFL...AN....VVK...A.FH-KAL....K...TLGIIMGTFTLCWLPFFI...VN....IVH...V.IQ-KAL....K...TTVILILAFFACWLPYYIgisID....SFIlleI.IKQG
KAT....Q...MLAIVLGVFIICWLPFFI...TH....ILN...I.HC-RATgragR...AMLAVAALYALCWGPHHA...L-....ILC...F.WY-G
KAT....V...TLAAVMGAFIICWFPYFT...AF....VYR...G.LR-G
RSF....L...TMFVVFVIFAICWAPLNC...IG....LAV...A.IN-P
VAN....QdpvSPSLVQG---------RI...VK....PNN...N.NM-P
RIT....R...MVLVVVGAFVVCWAPIHI...FV....IVW...T.LV-RIT....R...LVLVVVAVFVVCWTPIHI...FI....LVE...A.LGST
RIT....R...MVLVVVAVFIVCWTPIHI..yVI....IKA...L.VTIP
KTA....K...MLMVVLLVFALCYLPISV...LNvlkrVFG...M.FRQA
EVT....R...MVIIMVIAFLICWVPYAS...VA....FYI...F.THQG
KTV....K...TLGIIMGTFILCWLPFFI...VA....LVL...P.FCES
KVT....R...MVLVVVLVFAGCWLPFFT...VN....IVN...LaVALP
adenosine
...DCSHAPLW...LM...Y....LAIVLSHTNSVVNPFI-YAYRIREFRQTFRKIIRSHV-.LRQqepfkaagtsarvlaahgsdgeqvslrlnghppg
beta-1
...RE-LVPDR...LF...V....FFNWLGYANSAFNPII-Y-CRSPDFRKAFQGLLC---C.ARRaarrrhathgdrprasgclarpgpppspgaasdd
beta
...DN-LIRKE...VY...I....LLNWIGYVNSGFNPLI-Y-CRSPDFRIAFQELL----C.LRRsslkaygngyssngntgeqsgyhveqekenkllc
chemokine_r cefEN-TVHKW...I-...S....ITEALAFFHCCLNPIL-YAFLGAKFKTSAQHALTSVS-.-RGsslkilskgkrgghssvstesesssfhss.....
D2
...DC-NIPPV...LY...S....AFTWLGYVNSAVNPII-YTTFNIEFRKAFLKIL--HC-.---..................................
galanin
...RF-AFSPA...TYacrL....ASHCLAYANSCLNPLV-YALASRHFRARFRRLWP---C.GRRrrhrarralrrvrpassgppgcpgdarpsgrlla
histamine
...DD-AINEV...LE...A....IVLWLGYANSALNPIL-YAALNRDFRTGYQQLFC---CrLANrnshktslrsnasqlsrtqsreprqqeekplklq
melatonin
...QE-MAPQIpegLF...V....TSYLLAYFNSCLNAIV-YGLLNQNFRREYKRIL-----.LALwnprhciqdaskgshaeglqspappiigvqhqad
muscarinic
...S-SDDGLE...--...-....-HNKIQNGKAPRDPVTENCVQGEEKESSNDSTSVSAV-.ASNmrddeitqdentvstslghskdenskqtcirigt
delta
...DI-DRRDP...LV...VaalhLCIALGYANSSLNPVL-YAFLDENFKRCFRQLCR-KPC.GRPdpssfsrareatarervtactpsdgpgggaaa..
kappa
...SHSTAALS...SY...Y....FCIALGYTNSSLNPIL-YAFLDENFKRCFRDFCF---P.LKMrmerqstsrvrntvqdpaylrdidgmnkpv....
Mu-type
...ET-TFQTV...SW...H....FCIALGYTNSCLNPVL-YAFLDENFKRCFREFCIPTS-.-SNieqqnstrirqntrdhpstantvdrtnhqlenle
orexin
...SDREAVYA...CF...T....FSHWLVYANSAANPII-YNFLSGKFREQFKAAF----S.CCLpglgpcgslkapsprssashkslslqsrcsiski
rhodopsin
...SN--FGPI...FM...T....IPAFFAKSAAIYNPVIYI-MMNKQFRNCMLTTIC---C.GKNplgddeasatvsktetsqvapa............
5-hydroxytryp ...SC-HMPTL...LG...A....IINWLGYSNSLLNPVI-YAYFNKDFQNAFKKIIKCKF-.CRQ..................................
somatostatin ...QE-PASAG...LY...F....FVVILSYANSCANPVL-YGFLSDNFRQSFQKVL----C.LRKgsgakdadateprpdrirqqqeatrprtaaangl
TM7
Fig 19.Sequence aligment of many class A receptors based on hmm model
TM6
3. GPCRs
Nomenclature
Fig 20. Scheme of structure of the class A
A2A
B1AR
Rhodopsin
B2AR
----------------------------------IMGSSVY-----ITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITIS---TGFCAACHGCLFI
-----------------------------------------WEAGMSLLMALVVLLIVAGNVLVIAAIGSTQRLQTLTNLFITSLACADLVVGLLVVPFGATLVVRGTWL-WGSFLCELW
MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPW-QFSML-----AAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFV-FGPTGCNLE
--------------------------------DEV-WVVGM-----GIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWT-FGNFWCEFW
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
----------------------------------CCHHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHCGGGCCHHHHHHHHHHHHHHHHHHHHHHHHHHHH---CCCEEEHHHHHHH
-----------------------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHTTGGGCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCC -CCHHHHHHH
CCCEETTTTEECCCTTTTCCCTTTTTCCGGGCCHH-HHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHTTTTCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCTT-TTHHHHHHH
--------------------------------CHH-HHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHCGGGTTHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCTT-TTHHHHHHH
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
CFVLVLTQSSIFSLLAIAIDRYIAIR--IPLR-YN-GLVTGTRAKGIIAICWVLSFAIGL-TPMLGW-N-N-----CGQSQGCGEGQVA---C------LF-E-DVV------PMNYMV
TSLDVLCVTASIETLCVIAIDRYLAIT--SPFR-YQ-SLMTRARAKVIICTVWAISALVSFLPIMMHWWR-DEDPQAL---KCY----QDPGCC------DF-V---T------NRAYAGFFATLGGEIALWSLVVLAIERYVVVCKPMSNF--R---FGENHAIMGVAFTWVMALACAA-PPLVGWSRY---------------------IPEGMQCSCGIDYY-TPHEETNNESFVTSIDVLCVTASIETLCVIAVDRYFAIT--SPF-KYQSL-LTKNKARVIILMVWIVSGLTSFLPIQMHWYR-ATHQEAI---NCY----AEETCC------DF-F---T------NQAYA-
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
HHHHHHHHHHHHHHHHHHHHHHHHTTT--TTTT-TT-TTTTHHHHHHHHHHHHHHHHHHHH-GGGGTT-T-T-----TTCCTTTTTTEEE---T------TG-G-GCC------CHHHHH
HHHHHHHHHHHHHHHHHHHHHHHHHTT--THHH-HH-HHCCHHHHHHHHHHHHHHHHHHHHHHHHHTTTB-CCCHHHH---HHH----HTTTTT------CC-C---B------CHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHTTCTTTT--C---CCHHHHHHHHHHHHHHHHHHHH-HHHHTTTTE---------------------EEETTTCEEEETTT-TCTTTTTHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--TTT-TTTCC-CCHHHHHHHHHHHHHHHHHHHHHHHHHTTTB-CCCHHHH---HHH----HTTTTC------TT-T---B------CHHHH-
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
YFNFFACVLVPLLLMLGVYLRIFLAARR--QLNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYD
IASSIISFYIPLLIMIFVALRVYREAKEQI-----------------------------------------------------------------------------------------IYMFVVHFIIPLIVIFFCYGQLVFTVK--------------------------------------------------------------------------------------------IASSIVSFYVPLVIMVFVYSRVFQEAKRQ-LN----I-----------------------------------------------------------------------------------
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
HHHHHHHHHHHHHHHHHHHHHHHHHHCC--CCCHHHHHHHHHCCEEEEEETTTTCEEEETTEEEECCCCHHHHHHHHHHHHCCCTTTBCCHHHHHHHHHHHHHHHHHHHHHCCHHHHHHH
HHHHHHHHHHHHHHHHHHHHHHHHHHHHCC-----------------------------------------------------------------------------------------HHHHHHHCHHHHHHHHHHHHHHTTTTT--------------------------------------------------------------------------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-CC----H-----------------------------------------------------------------------------------
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
SLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRW--------------------------DEAAVNLA---------K--SRWYN-------QTPNRAKRVITTFR---TG----------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------- ------------------------------------------------FEMLRIDEGLRLKIYKDTEGYYTIGIG--HL--LTKSPSLNAAKSELDKAIGRNTNGVIT-KDEAEKLFNQDVDAAVRGILRN
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
HCCHHHHHHHHHHHHHHCHHHHHTTHHHHHHHHHCCH--------------------------HHHHHHHH---------C--CHHHH-------HCHHHHHHHHHHHH---HC----------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------- ------------------------------------------------HHHHHHHHCCEEEEEETTTTCEEEETT--EE--EECCCCHHHHHHHHHHHHCCCTTTBCC-HHHHHHHHHHHHHHHHHHHHHT
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
--------------TW---DAYR-----------------------------------------------------------S------TL-QKEVHAAKSLAIIVGLFALCWLPLHIIN
--------------------------------------------------------------------------------------------- REHKALKTLGIIMGVFTLCWLPFFLVN
--------------------------------------------------------------------------------E--AAASATTQ-KAEKEVTRMVIIMVIAFLICWLPYAGVA
AKLKPVYDSLDAVRRAALIN---MVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKF------CLKEHKALKTLGIIMGTFTLCWLPFFIVN
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
--------------CH---HHHH-----------------------------------------------------------H------HH-HHHCHHHHHHHHHHHHHHHHHHHHHHHH
--------------------------------------------------------------------------------------------- HHHHHHHHHHHHHHHHHHHHHHHHHHH
--------------------------------------------------------------------------------T--TCCCCCCH-HHHHHHHHHHHHHHHHHHHHHHHHHHHH
TTHHHHHHHCCHHHHHHHHH---HHHHHHHHHHHHCHHHHHHHHHCCHHHHHHHHHCCHHHHHCHHHHHHHHHHHHHCCCGGGTT------TCHHHHHHHHHHHHHHHHHHHHHHHHHHH
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
CFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLR --Q---------------IVNVFNRDL--VPDWLFVAFNWLGYANSAMNPIIYC-RSPDFRKAFKRLLA-----------------------FYIFTHQGSD-FGPIFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCC----GKNPSTTVSKTETSQVAPA
IVHVIQDNL--IRKEVYILLNWIGYVNSGFNPLIYC-RSPDFRIAFQELLC-------L----------------
ClassA_stamp_VMDA2A
ClassA_stamp_VMDAvi
ClassA_stamp_VMDBov
ClassA_stamp_VMDHum
HHHHHTTTTCCCCHHHHHHHHHHHHHHCHHHHHHHHHHCHHHHHHHHHHHHHHHCC --C---------------HHHHHTTTT--CCHHHHHHHHHHHHHHHHHHHHHHH-HCHHHHHHHHHHHC-----------------------HHHHHTTTTC-CCHHHHHHHHHHHGGGGHHHHHHHHHHCHHHHHHHHHHHHT----TTCCCCCCTTTTTTCCCCC
HHHHHTTTT--TTHHHHHHHHHHHHHHHCHHHHHHH-CCHHHHHHHHHHHC-------C----------------
TM1
TM3
TM2
TM4
TM5
Lysozyme
Lysozyme
Lysozyme
TM7
Fig 21. Structural alignment of class A receptors
TM6
3. GPCRs
Structural Conservation
SCORE 5.57
RMSD 1.92
Fig22. Superimposition of β2AR, β1AR, A2AR and Rhodopsin
Fig23. Superimposition of β2AR, β1AR, A2AR and Rhodopsin
3. GPCRs
Structural Conservation
Fig24. Superimposition of β2AR, β1AR, A2AR and Rhodopsin
3. GPCRs
β2 Adrenergic Receptor
>beta 2 adrenergic receptor [Homo sapiens]
MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKF
ERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIET
LCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYA
NETCCDFFTNQAYAIASSIVSFYVPLVIMAFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVE
QDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNW
IGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKL
LCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL
Fig25. β2 Adrenergic Receptor sequence
β2AR
G proteins
Adenilate
cyclase
AMPc
PKA
Smooth muscle relaxation, blood vessels dilatation, striated muscle contraction,
bronchiole dilation...
• The first non-rhodopsin GPCR cloned
• One of the most extensively studied members of this large receptor family.
3. GPCRs
β2 Adrenergic Receptor
MEMSAT software to
predict alpha helix
transmembrane
regions based on Dr.
Jones
predictive
algorithm described
in 1994
Fig26 Hydropathy plot of β2 Adrenergic Receptor sequence
Fig27. Hydropathy plot of β2 Adrenergic Receptor sequence
3. GPCRs
β2 Adrenergic Receptor
ECL2
Extracellular
II
VI
IV VII
III
I
carazolol
V
VIII
T4-lysozyme
Fig28. β2 Adrenergic Receptor
Intracellular
3. GPCRs
β2 Adrenergic Receptor
TM1
TM2
TM3
TM4
TM5
TM6
TM7
Fig29. Sequence and secondary structure of β2
Adrenergic Receptor sequence obtained from PDB
3. GPCRs
β2 Adrenergic Receptor
ECL2
Extracellular
II
VI
IV VII
III
I
carazolol
V
VIII
T4-lysozyme
Fig30. β2 Adrenergic Receptor
Intracellular
3. GPCRs
ECL2 (Extracellular Loop 2)
ECL2
Cys 190
Cys 184
Cys 191
Cys 106
Phe 193
Carazolol
Fig31. Extracellular Loop 2 of the β2 Adrenergic Receptor
3. GPCRs
ECL2 (Extracellular Loop 2)
ECL2
Cys 190
Rhodopsin
Cys 184
Cys 191
Cys 106
ECL2
Phe 193
Cys 187
Cys 110
Carazolol
Fig32. Extracellular Loop 2 of the β2 Adrenergic Receptor
β2-Adrenergic Receptor
Retinal
Fig33. Extracellular Loop 2 of Rhodopsin
3. GPCRs
ECL2 (Extracellular Loop 2)
ECL2
Cys 159
Cys 71
Cys 146
ECL1
Cys 166
Cys 74
Cys 77
Fig34. Extracellular Loop 2 of β2-Adrenergic Receptor, Rhodopsin and
A2- Adenosin Receptor with the ligands carazolol and retinal
Fig36. Extracellular Loop 2 of β2-Adrenergic
Fig35.
A2- Adenosin Receptor
Receptor,with
Rhodopsin
its
three disulfur
and A2bonds.
Adrenergic Receptor with their
conserved disulfur bonds.
3. GPCRs
β2 Adrenergic Receptor
ECL2
Extracellular
II
VI
IV VII
III
I
carazolol
V
VIII
T4-lysozyme
Fig37. β2 Adrenergic Receptor
Intracellular
3. GPCRs
Ligand binding site
Fig38. Extracellular view of the ligand binding site of the β2 Adrenergic Receptor with its ligand carazolol
3. GPCRs
Ligand binding site
Carazolol
Ser203
Ser203
Asp113Asn312
Ser203
Ser203
Asp113
Carazolol
Asn312
Fig39. Interaction between β2-Adrenergic Receptor and carazolol
Fig40. Interaction between β2-Adrenergic Receptor and carazolol
3. GPCRs
Ligand binding site
Trp109
Trp109
Tyr199
Phe193
Val114
Trp286
Phe193
Val114
Phe289
Phe290
Phe289
Tyr199
Phe290
Trp286
Fig42. Interaction
Fig41. Interaction
between
between
β2-Adrenergic
β2-Adrenergic
Receptor
Receptor
and carazolol
and carazolol
3. GPCRs
Ligand binding site
Fig43. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin
and retinal
3. GPCRs
Ligand binding site
Fig44. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin and retinal
3. GPCRs
Ligand binding site
Phe5.47
Tyr6.51
Tyr6.51
Trp6.48
Phe5.47
Trp6.48
Fig45. Superimposition of the ligand binding site of Rhodopsin
and β2-Adrenergic Receptor
Fig46. Superimposition of the ligand binding site of Rhodopsin
and β2-Adrenergic Receptor
3. GPCRs
Ligand binding site
Rhodopsin
β2-Adrenergic Receptor
Trp6.48
Trp6.48
Fig47. Interaction between Rhodopsin and retinal through Trp6.48
Fig48. Interaction between β2-Adrenergic Receptor and carazolol and
Trp6.48
“Toogle Switch”
3. GPCRs
β2 Adrenergic Receptor
ECL2
Extracellular
II
VI
IV VII
III
I
carazolol
V
VIII
T4-lysozyme
Fig49. β2 Adrenergic Receptor
Intracellular
3. GPCRs
Ionic Lock
Rhodopsin
β2-Adrenergic Receptor
Tyr3.51
Tyr3.51
Tyr3.51
Asp3.49
Glu/Asp3.49
Arg3.50
Arg3.50
Arg3.50
Glu3.49
Glu6.30
Glu6.30
Fig50. Ionic lock of Rhodopsin
Glu6.30
Fig51. Ionic lock of β2-Adrenergic Receptor
Conserved E/D(E)RY motif
Fig52. Superimposition of the Ionic lock of Rhodopsin and β2-Adrenergic Receptor
3. GPCRs
Activation
ECL2
ECL2
II
VI
IV VII
Extracellular
Extracellular
III
II
IV
I
carazolol
V
BI-167107
V
VIII
I
VII
VIII
III
VI
T4-lysozyme
Fig53. β2 Adrenergic Receptor
Nanobody (Nb80)
Intracellular
Intracellular
Fig54. Nanobody stabilized-β2 Adrenergic Receptor
3. GPCRs
Activation
Fig55. Conserved prolines in β2 Adrenergic Receptor
3. GPCRs
Activation
I
ECL2
II
VII III
IV
I
II
IV
V
ICL2
VI
III
VII
V
VI
Fig56. Superimposition of β2 Adrenergic Receptor in its
activated and inactivated states.
Fig57. Superimposition of β2 Adrenergic Receptor in its activated and
inactivated states.
3. GPCRs
Activation
Tyr199
Phe193
Phe193
Trp109
Tyr199
Val114
Val114
Phe290
Phe290
Trp286
Phe289
Ile309
Fig58. Interaction between β2-Adrenergic Receptor and BI-167107
Trp286
Phe289
Fig59. Interaction between β2-Adrenergic Receptor and carazolol
3. GPCRs
Activation
Carazolol
BI-167107
Ser203
Ser203
Asp113
Asp113 BI-167107
Ser203
Asn312
Asn312
Asn312
Ser207
Fig60. Interaction between β2-Adrenergic Receptor and BI-167107
Fig61. Interaction between β2-Adrenergic Receptor and carazolol
Fig62. Interaction between β2-Adrenergic Receptor and BI-167107
3. GPCRs
Activation
Ser2075.46
Ser203
BI-167107
carazolol
Ser207
Ser203
Pro2115.50
Ser207
Ile1213.40
Ile121
Asn318
Asn3187.45
Pro2115.50
Ile1213.40
Pro211
Phe2826.44
Asn3187.45
Phe282
Fig64. Adrenergic Receptor in its activated state.
Fig63. β2 Adrenergic Receptor in its inactivated state.
Phe2826.44
Phe2826.44
Fig65. Superimposition of β2 Adrenergic Receptor in its activated and inactivated
states.
3. GPCRs
Activation
Trp6.48
Fig66. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
3. GPCRs
Activation
Glu268
Arg131
Fig67. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
3. GPCRs
Activation
Glu268
Arg131
Fig68. β2 Adrenergic Receptor coupled to G-protein
4.Conclusions
G-protein-coupled
receptors represent the
largest class of drug
targets, and its structure
determination is very
important to design new
specific drugs.
Although the
crystallization methods
have improved incredibly,
we need to crystallize
new states of structures
that have already been
crystallized to understand
better GPCR dynamics
such as the activation
process.
In addition, the
crystallization of new
structures is needed to
amplify the
knowledge of these
receptors and to
design new drugs.
5. Bibliography
Costanzi S. On the Applicability of GPCR Homology Models to Computer-Aided Drug Discovery: A
Comparison between In Silico and Crystal Structures of the β2- Adrenergic receptor. J. Med.Chem. 2008;
51: 2907-2914.
Cherezov V, Rosenbaum D.M, Hanson M.A, Rasmussen S.G.F, Thian F.S, Kobilka T.S., Choi H, Kuhn P, Weis
W.I, Kobilka B.K, Stevents R.C. High – Resolution Crystal Structure of an Engineered Human β2Adrenergic G Protein – Coupled Receptor.
Deupi X, Kobilka B.K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function.
Physiology (Bethesda). 2010: 25; 293–303
Deupi X, Standfuss J. Structural insights into agonist-induced activation of G-protein-coupled receptors.
Current Opinion in Structural Biology. 2011; 21: 541 – 551.
Dror R. O, Arlow D. H, Maragakis P, Mildorf T. J, Pan A. C, Xu H, Borhani D. W, Shaw D. E. Activation
mechanism of the β2 - Adrenergic receptor. PNAS. 2011; 108: 18684 – 18689.
Fairman J.W, Noinaj N, Buchanan S.K. The structural biology of β- barrel membrane proteins: a summary
of recent report. Current Opinion Structural Biology. 2011; 21: 523 – 531.
Goetz A, Lanig, Gmeiner P, Clark T. Molecular Dynamics Simulations of the Effect of the G-Protein and
Diffusible Ligands β2-Adrenergic Receptors. J. Mol.Biol. 2011; 414: 611 – 623.
González A, Perez-Acle T, Pardo L, Deupi X. Molecular Basis of Ligand Dissociation in β-Adrenergic
Receptor. Plos ONE. 2011; 6.
5. Bibliography
Jones D.T., Taylor W.R., Thornton J.M. A model recognition Approach to the prediction of All – Helical
Membrane Protein Structure and Toxicology. Biochemistry. 1994; 33 (10): 3038-3049.
Kahsai A. W, Xiao K, Rajagopal S, Ahn S, Shukla A, Sun J, Oas T. G, Lefkowitz R. J. Multiple ligand – specific
conformations of the β2- adrenergic receptor. Nature Chemical Biology. 2011; 7: 692 – 700.
Katritch V, Abagyan R. GPCR agonist binding revealed by modeling and crystallography. Trends in
Pharmacological Science. 2011; 31 (11): 637 – 643.
Kobilka B.K. Structural insights into andrenergic receptor function and pharmacology. Trends in
Pharmacological Science. 2011; 21 (4): 213-218.
Kolb P, et al. Structure-based discovery of β2- Adrenergic receptor ligands. Proc. Natl. Acad. Sci. U.S.A.
2009; 106: 6843–6848López Muñoz L. Homology modeling and structural analysis of the antipsychotic drugs receptorome.
[Doctoral Thesis]. Universitat Pompeu Fabra; 2010.
Massotte D, Kieffler B. L. Structure – Function Relationships in G Proteins – Coupled Receptors
Handbook. In: Devi L. A, editor. The G Protein – Coupled Receptors. New Yersey: Humana Press; 2005. 339.
Palczewski K, et al. Crystal structure of rhodopsin: a G protein-Coupled receptor. Science. 2000; 289: 739–
745.
5. Bibliography
Rasmussen S.G, et al. Crystal structure of the human β2 -Adrenergic G-protein-coupled receptor. Nature.
2007; 450: 383–387.
Rasmussen S.G, DeVree B.T, Zou Y., Kruse A.C, Chung K.Y, Kobilka T.S. Crystal structure of the β2
Adrenergic Receptor-Gs protein complex. Nature. 2011; 477: 549-555.
Rasmussen S.G, et al. Structure of a nanobody-stabilized active state of the β2 - adrenoceptor. Nature.
2011; 469: 175–180.
Rosenbaum D.M,et al. GPCR engineering yields high-resolution structural insights into β2 -adrenergic
receptor function. Science. 2007; 318: 1266–1273
Rosenbaum D. M, Rasmussen S. G, Kobilka B.K. The β2- Adrenergic Receptor as a Model for G-ProteinCoupled Receptor Structure and Activation by Diffusible Hormones. Handbook of Cell Signaling. 2010;
Chapter 25: 163-168.
Rosenbaum D.M, et al. Structure and function of an irreversible agonist-β2 adrenoceptor complex.
Nature. 2011; 469: 236–240.
Sabio M, et al. Use of the X-ray structure of the β2- Adrenergic receptor for drug discovery. Part 2:
identification of active compounds. Bioorg. Med. Chem. Lett. 2008; 18: 5391–5395.
Sansuk K, et al. A structural insight into the reorientation of transmembrane domains 3 and 5 during
family A GPCR activation. Mol. Pharmacol. 2011; 79: 262–269
5. Bibliography
Schulz G. β- barrel membrane proteins. Current Opinion in Structural Biology. 2000; 10: 443 – 447.
Topiol S, Sabio M. X-ray structure breakthroghs in the GPCR transmembrane region. Biochemical
Pharmacology. 2009; 79: 11 – 20.
Warne T, et al. Structure of a β1- Adrenergic G-protein-coupled receptor. Nature. 2008; 454: 486–491.
Weis W. I, Kobilka B. K. Structural insights into G-protein-coupled receptor activation. Current Opinion
Structural Biology. 2008; 18: 734 – 740.
Wimley W.C. The versatile β- barrel membrane protein. Current Opinion in Structural Biology. 2003; 13:
404 – 411.
Worth C. L., Kleinau G, Krause G. Comparative Sequence and Structural Analysen of G-protein-coupled
Receptor Crystal Structures and Implications for Molecular Models. Plos One. 2009; 9.
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