Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS.
-
Upload
phebe-cole -
Category
Documents
-
view
236 -
download
1
Transcript of Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE 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. Conclusions5. 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
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.
Membrane Proteins:
Extracellular region
Intracellular region
Transmembrane region
Transmembrane proteins
Domain Functions
Extracellular Involved in cell – cell signalingLet interactions with other molecules
Transmembrane Forms channels and pores
Intracellular Activates intracellular signaling pathways
1. Introduction
Fig3. Cell membrane with a transmembrane protein.
CRYSTALLIZATION. Why is it so complicated?
Membrane protein structural biology:
The most challenging targets in structural biology
1. Introduction
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
CRYSTALLIZATION. Why is it so complicated?
Trying to solubilize and crystalize transmembrane proteins:
1. Introduction
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
To solubilize transmembrane proteins is necessary to add amphipathic molecules such as detergents to cover the transmembrane region
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. Conclusions5. 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
Fig4. NalP protein Fig5. OmpA protein
Extracellular
Intracellular
Fig6. NalP protein Fig7. OmpA protein
Extracellular
Intracellular
2. Types of transmembrane proteins
α - Helical
Extracellular
Fig9. Rodhopsin
Intracellular
Fig8. PufX protein
•Contain a hydrophilic domain on each side of the membrane
Bitopic
•Contain a multiple hydrophilic domains on both sides of the membrane
Polytopic
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. Conclusions5. 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
One of the most important types of receptors
GPCRThe most important family of membrane proteins
3. GPCRs Classification
Fig13. Scheme of structure of the class C
Fig12. Scheme of structure of the class B
Fig11. Scheme of structure of the class A
3. GPCRsSequence Conservation
A_adenosine P..FAI..TI.........---.........STGFCA..ACHGCLF.IACFVLVLTQSSIFSLLA..IA..IDRY..IA.....I--RIPL-RY-NGLVT A_beta-1 P..FGA..TI.........VVW.........GRW-EY..GSFFCEL.WTSVDVLCVTASIETLCV..IA..LDRY..LA.....I--TSPF-RY-QSLLT A_beta P..FGA..AH.........ILM.........KMW-TF..GNFWCEF.WTSIDVLCVTASIETLCV..IA..VDRY..FA.....I--TSPF-KY-QSLLT A_rhodopsin T..STL..YT.........SLH.........GYF-VF..GPTGCNL.EGFFATLGGEIALWSLVV..LA..IERY..VV.....VCKPMSN---F--RFG B_Glucagon R..YSQ..KIg......ddLSV.........STW-LSdgAVAGCRV.AAVFMQYGIVANYCWLLVegLY..LHNL..LG.....L-ATLPERSFF----- B_Pituitary A..ISV..FI.........---.........KDWILY..AEQDSN-.HCFISTVECKAVMVFFHY..CV..VSNY..FW.....L-FIEGL--YLFTLLV B_Parathyroid Y..SGA..TLdeaerlteeELRaiaqappppATA-AA..GYAGCRVaVTFFLYFLATNYYWILVE..--..-GLY..LH.....SLIFMAF---FS---E B_Vasoactive IkdLAL..FD.........SGE.........SDQCSE..GSVGCKA.AMVFFQYCVMANFFWLLV..EG..LYLYtlLA.....V-SFFSERKYF----- C_Gamma-aminob P..LGLdgYH.........IGR.........NQF---..-PFVCQ-.-ARLWLLGLGFSLG----..--..---Y..GS.....MFTKIWW---VHTVFT C_Metabotropic P..FTL..IA.........--K.........PTT---..--TSCYL.QRLLVGLSSAMCYSALVT..KTnrIARI..LAgskkkICTRKP-------RFM C_Metabotropic P..STA..VC.........TLR.........RLG---..--LGTAF.SVCYSALLTKTN------..-R..IARI..FG.....-GAREGA---QRPRFI C_Taste P..TRP..AC.........LLR.........QALFAL..G-----F.TIFLSCLTVRS--FQLII..I-..----..FK.....FSTKVPTF--YHAWVQ
A_adenosine GTRAKGIIAIC...WVL.SFAIG-.LTPM....LGW-N----NCGQPKEGKNHSQGCGEGQVAC.LF-EDV--.-----VPMNYMVYFN..FFA.CVLVP A_beta-1 RARARGLVCTV...WAI.SALVSF.LPIL....MHWWRAESDEA--RR--CYNDPKC------C.D-F--VT-.------NRAY-AIAS..SVV.SFYVP A_beta KNKARVIILMV...WIV.SGLTSF.LPIQ....MHWYRATHQEA--IN--CYANETC------C.D-F--FT-.------NQAY-AIAS..SIV.SFYVP A_rhodopsin ENHAIMGVAFT...WVM.ALACAA.-PPL....AGWS-------------R---YIPEGLQCSC.GID--YYT.LKPEVNNESF-VIYM..FVV.HFTIP B_Glucagon -----------...---.SLYLG-.----....IGW------GAPMLFVVPWAVVK-------C.LF-ENV-Q.CWTSNDNMGFWWILR..FPV.FLAI- B_Pituitary ETFFPERRYFY...WYT.IIGWGT.PTVC....VTVW------A-TLR--LYFDDTG------CwDM-NDST-.------ALWW-VIKG..PVVgSIMVN B_Parathyroid KKYLWGFTVFG...WGLpAVFVAV.----....--WV---SVRA------TLANTG-------C.W-D--LS-.----SGNKKW-IIQV..PIL.ASIV- B_Vasoactive ----WGYILIG...WGV.PSTFT-.----....MVWT-----IA-RIH---FEDYG-------C.W-D---T-.-----INSSLWWIIKgpILT.SILVN C_Gamma-aminob KKEEKKEWRKTlepWKL.YATVGL.LVGMdvltLAIWQIVDPLHRTIE--TFAKEEP-----KE.DID--VSIlPQLEHCSSRKMNTWL..GIF.YGYKG C_Metabotropic SAWAQVIIA--...---.SILISVqLTLV....VTLI---------IM--E--PPMP-------.-IL--SY-.---PSIKEVYLICNT..SNL.GVVAP C_Metabotropic SPASQVAIC--...--L.ALISGQ.LLIV....VAWL---VVEA----PGTGKETAP-------.ER-REVV-.----TLRCNH-RDAS..MLG.SLAYN C_Taste 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
Fig17. Scheme of structure of the class C
Fig16. Scheme of structure of the class B
Fig15. Scheme of structure of the class A
Class A. SCOP
Fig 18. Class A classification from SCOP (Structural Classification of Proteins)
3. GPCRs
Class A. Chronology
Before 2000
2000
2007
2008
2008-2012
RhodopsinPalczewski et al. Aug. 2000
β1 -Adrenergic Receptor Warne et al. July 2008
2-Adenosine Receptor Jaakola et al. Oct. 2008β2-Adrenergic
Receptor
D1-RH1-RSPS1…
3. GPCRs
Cherezov et al. Nov. 2007
TM7
TM6TM5
TM4TM3TM2TM1
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-ANVSD2 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--TRPkappa FVVGLVGNSLVMFVIIR..YTKMKTA...TNIYIFNLALAD-ALVTTTMP.FQSTVYLMNSW-PFGDVLCKIVISIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALD-FR---.TPLK.A.KIINICIWLLSSSVGISAIVLGG-TKVREMu-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
adenosine NCGQP.KEGKNHSQGCGEGQVACLF-EDV-------VPMNYMVYFNF...FACVLVPLLLMLGVYLR....I...FLAAR... HAA....K...SLAIIVGLFALCWLPLHI...IN....CFT...F.FC-Pbeta-1 EA--R.R--CYNDPKC------CD-F--VT-------NRAY-AIASS...VVSFYVPLCIMAFVYLR....V...FREAQ... KAL....K...TLGIIMGVFTLCWLPFFL...AN....VVK...A.FH--beta EA--I.N--CYANETC------CD-F--FT-------NQAY-AIASS...IVSFYVPLVIMAFVYSR....V...FQEAK... KAL....K...TLGIIMGTFTLCWLPFFI...VN....IVH...V.IQ--chemokine_r EA--D.D--R---YI-------CDRF--YP-------NDLWVVVFQFqhiMVGLILPGIVILSCYCI....I...ISKL-... KAL....K...TTVILILAFFACWLPYYIgisID....SFIlleI.IKQGD2 NA---.---DQNE---------CIIA-----------NPAF-VVYSS...IVSFYVPFIVTLLVYIK....I...YIVLR... KAT....Q...MLAIVLGVFIICWLPFFI...TH....ILN...I.HC--galanin --GTV.R--YGALEL-------CV-P--AW---EDARRRAL-DVATF...AAGYLLPVAVVSLAYGRtlrfL...WAAVG... RATgragR...AMLAVAALYALCWGPHHA...L-....ILC...F.WY-Ghistamine TS---.---KGNHTTS-----KCKVQ--V--------NEVY-GLVDG...LVTFYLPLLIMCITYYR....I...FKVAR... KAT....V...TLAAVMGAFIICWFPYFT...AF....VYR...G.LR-Gmelatonin -----.---EYDPRIY-----SCT-F--IQ-----TASTQY-TAAVV...VIHFLLPIAVVSFCYLR....IwvlVLQAR... RSF....L...TMFVVFVIFAICWAPLNC...IG....LAV...A.IN-Pmuscarinic QF--I.VGVRTVEDGE------CYIQ--FF------SNAAV-TFGTA...IAAFYLPVIIMTVLYWH....I...SRASK... VAN....QdpvSPSLVQG---------RI...VK....PNN...N.NM-Pdelta RDGAV.VCML-QFPSP-----SWYWD-TVT------------KICVF...LFAFVVPILIITVCYGL....M...LLRLR... RIT....R...MVLVVVGAFVVCWAPIHI...FV....IVW...T.LV--kappa DVDVI.ECSL---QFP-------D-D-DYS------WWDLFMKICVF...IFAFVIPVLIIIVCYTL....M...ILRLK... RIT....R...LVLVVVAVFVVCWTPIHI...FI....LVE...A.LGSTMu-type ----T.K--YRQGSID------CTLT--FS--HPTWYWENLLKICVF...IFAFIMPVLIITVCYGL....M...ILRLK... RIT....R...MVLVVVAVFIVCWTPIHI..yVI....IKA...L.VTIPorexin ECSSVlP--ELANRTR--LFSVCD--ERWA---DDLYPKIY-HSCFF...IVTYLAPLGLMAMAYFQ....I...FRKLW... KTA....K...MLMVVLLVFALCYLPISV...LNvlkrVFG...M.FRQArhodopsin -----.---R---YIPEGLQCSCGID--YYTLKPEVNNESF-VIYMF...VVHFTIPMIIIFFCYGQ....L...VFTVK... EVT....R...MVIIMVIAFLICWVPYAS...VA....FYI...F.THQG5-hydroxytryp ED---.---R-SDPDA------CTISKDH----------GY-TIYST...FGAFYIPLLLMLVLYGR....I...FRAARfri KTV....K...TLGIIMGTFILCWLPFFI...VA....LVL...P.FCESsomatostatin EGGTC.NASW---PEP------VGLW-----------GAVF-IIYTA...VLGFFAPLLVICLCYLL....I...VVKVR... KVT....R...MVLVVVLVFAGCWLPFFT...VN....IVN...LaVALP
adenosine ...DCSHAPLW...LM...Y....LAIVLSHTNSVVNPFI-YAYRIREFRQTFRKIIRSHV-.LRQqepfkaagtsarvlaahgsdgeqvslrlnghppgbeta-1 ...RE-LVPDR...LF...V....FFNWLGYANSAFNPII-Y-CRSPDFRKAFQGLLC---C.ARRaarrrhathgdrprasgclarpgpppspgaasddbeta ...DN-LIRKE...VY...I....LLNWIGYVNSGFNPLI-Y-CRSPDFRIAFQELL----C.LRRsslkaygngyssngntgeqsgyhveqekenkllcchemokine_r cefEN-TVHKW...I-...S....ITEALAFFHCCLNPIL-YAFLGAKFKTSAQHALTSVS-.-RGsslkilskgkrgghssvstesesssfhss.....D2 ...DC-NIPPV...LY...S....AFTWLGYVNSAVNPII-YTTFNIEFRKAFLKIL--HC-.---..................................galanin ...RF-AFSPA...TYacrL....ASHCLAYANSCLNPLV-YALASRHFRARFRRLWP---C.GRRrrhrarralrrvrpassgppgcpgdarpsgrllahistamine ...DD-AINEV...LE...A....IVLWLGYANSALNPIL-YAALNRDFRTGYQQLFC---CrLANrnshktslrsnasqlsrtqsreprqqeekplklqmelatonin ...QE-MAPQIpegLF...V....TSYLLAYFNSCLNAIV-YGLLNQNFRREYKRIL-----.LALwnprhciqdaskgshaeglqspappiigvqhqadmuscarinic ...S-SDDGLE...--...-....-HNKIQNGKAPRDPVTENCVQGEEKESSNDSTSVSAV-.ASNmrddeitqdentvstslghskdenskqtcirigtdelta ...DI-DRRDP...LV...VaalhLCIALGYANSSLNPVL-YAFLDENFKRCFRQLCR-KPC.GRPdpssfsrareatarervtactpsdgpgggaaa..kappa ...SHSTAALS...SY...Y....FCIALGYTNSSLNPIL-YAFLDENFKRCFRDFCF---P.LKMrmerqstsrvrntvqdpaylrdidgmnkpv....Mu-type ...ET-TFQTV...SW...H....FCIALGYTNSCLNPVL-YAFLDENFKRCFREFCIPTS-.-SNieqqnstrirqntrdhpstantvdrtnhqlenleorexin ...SDREAVYA...CF...T....FSHWLVYANSAANPII-YNFLSGKFREQFKAAF----S.CCLpglgpcgslkapsprssashkslslqsrcsiskirhodopsin ...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
3. GPCRs
Fig 19.Sequence aligment of many class A receptors based on hmm model
Lysozyme
Lysozyme
Lysozyme
TM1 TM2
TM3 TM4
TM5
TM6
TM7
A2A ----------------------------------IMGSSVY-----ITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITIS---TGFCAACHGCLFI B1AR -----------------------------------------WEAGMSLLMALVVLLIVAGNVLVIAAIGSTQRLQTLTNLFITSLACADLVVGLLVVPFGATLVVRGTWL-WGSFLCELW Rhodopsin MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPW-QFSML-----AAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFV-FGPTGCNLE B2AR --------------------------------DEV-WVVGM-----GIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWT-FGNFWCEFW
ClassA_stamp_VMDA2A ----------------------------------CCHHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHCGGGCCHHHHHHHHHHHHHHHHHHHHHHHHHHHH---CCCEEEHHHHHHH ClassA_stamp_VMDAvi -----------------------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHTTGGGCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCC-CCHHHHHHH ClassA_stamp_VMDBov CCCEETTTTEECCCTTTTCCCTTTTTCCGGGCCHH-HHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHTTTTCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCTT-TTHHHHHHH ClassA_stamp_VMDHum --------------------------------CHH-HHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHCGGGTTHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCTT-TTHHHHHHH
ClassA_stamp_VMDA2A CFVLVLTQSSIFSLLAIAIDRYIAIR--IPLR-YN-GLVTGTRAKGIIAICWVLSFAIGL-TPMLGW-N-N-----CGQSQGCGEGQVA---C------LF-E-DVV------PMNYMV ClassA_stamp_VMDAvi TSLDVLCVTASIETLCVIAIDRYLAIT--SPFR-YQ-SLMTRARAKVIICTVWAISALVSFLPIMMHWWR-DEDPQAL---KCY----QDPGCC------DF-V---T------NRAYA- ClassA_stamp_VMDBov GFFATLGGEIALWSLVVLAIERYVVVCKPMSNF--R---FGENHAIMGVAFTWVMALACAA-PPLVGWSRY---------------------IPEGMQCSCGIDYY-TPHEETNNESFV- ClassA_stamp_VMDHum TSIDVLCVTASIETLCVIAVDRYFAIT--SPF-KYQSL-LTKNKARVIILMVWIVSGLTSFLPIQMHWYR-ATHQEAI---NCY----AEETCC------DF-F---T------NQAYA-
ClassA_stamp_VMDA2A HHHHHHHHHHHHHHHHHHHHHHHHTTT--TTTT-TT-TTTTHHHHHHHHHHHHHHHHHHHH-GGGGTT-T-T-----TTCCTTTTTTEEE---T------TG-G-GCC------CHHHHH ClassA_stamp_VMDAvi HHHHHHHHHHHHHHHHHHHHHHHHHTT--THHH-HH-HHCCHHHHHHHHHHHHHHHHHHHHHHHHHTTTB-CCCHHHH---HHH----HTTTTT------CC-C---B------CHHHH- ClassA_stamp_VMDBov HHHHHHHHHHHHHHHHHHHHHHHHHHTTCTTTT--C---CCHHHHHHHHHHHHHHHHHHHH-HHHHTTTTE---------------------EEETTTCEEEETTT-TCTTTTTHHHHH- ClassA_stamp_VMDHum HHHHHHHHHHHHHHHHHHHHHHHHHHH--TTT-TTTCC-CCHHHHHHHHHHHHHHHHHHHHHHHHHTTTB-CCCHHHH---HHH----HTTTTC------TT-T---B------CHHHH-
ClassA_stamp_VMDA2A YFNFFACVLVPLLLMLGVYLRIFLAARR--QLNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYD ClassA_stamp_VMDAvi IASSIISFYIPLLIMIFVALRVYREAKEQI------------------------------------------------------------------------------------------ ClassA_stamp_VMDBov IYMFVVHFIIPLIVIFFCYGQLVFTVK--------------------------------------------------------------------------------------------- ClassA_stamp_VMDHum IASSIVSFYVPLVIMVFVYSRVFQEAKRQ-LN----I-----------------------------------------------------------------------------------
ClassA_stamp_VMDA2A HHHHHHHHHHHHHHHHHHHHHHHHHHCC--CCCHHHHHHHHHCCEEEEEETTTTCEEEETTEEEECCCCHHHHHHHHHHHHCCCTTTBCCHHHHHHHHHHHHHHHHHHHHHCCHHHHHHH ClassA_stamp_VMDAvi HHHHHHHHHHHHHHHHHHHHHHHHHHHHCC------------------------------------------------------------------------------------------ ClassA_stamp_VMDBov HHHHHHHCHHHHHHHHHHHHHHTTTTT--------------------------------------------------------------------------------------------- ClassA_stamp_VMDHum HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-CC----H-----------------------------------------------------------------------------------
ClassA_stamp_VMDA2A SLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRW--------------------------DEAAVNLA---------K--SRWYN-------QTPNRAKRVITTFR---TG------ ClassA_stamp_VMDAvi ------------------------------------------------------------------------------------------------------------------------ ClassA_stamp_VMDBov ------------------------------------------------------------------------------------------------------------------------ ClassA_stamp_VMDHum -------------------------------------FEMLRIDEGLRLKIYKDTEGYYTIGIG--HL--LTKSPSLNAAKSELDKAIGRNTNGVIT-KDEAEKLFNQDVDAAVRGILRN
ClassA_stamp_VMDA2A HCCHHHHHHHHHHHHHHCHHHHHTTHHHHHHHHHCCH--------------------------HHHHHHHH---------C--CHHHH-------HCHHHHHHHHHHHH---HC------ ClassA_stamp_VMDAvi ------------------------------------------------------------------------------------------------------------------------ ClassA_stamp_VMDBov ------------------------------------------------------------------------------------------------------------------------ ClassA_stamp_VMDHum -------------------------------------HHHHHHHHCCEEEEEETTTTCEEEETT--EE--EECCCCHHHHHHHHHHHHCCCTTTBCC-HHHHHHHHHHHHHHHHHHHHHT
ClassA_stamp_VMDA2A --------------TW---DAYR-----------------------------------------------------------S------TL-QKEVHAAKSLAIIVGLFALCWLPLHIIN ClassA_stamp_VMDAvi ---------------------------------------------------------------------------------------------REHKALKTLGIIMGVFTLCWLPFFLVN ClassA_stamp_VMDBov --------------------------------------------------------------------------------E--AAASATTQ-KAEKEVTRMVIIMVIAFLICWLPYAGVA ClassA_stamp_VMDHum AKLKPVYDSLDAVRRAALIN---MVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKF------CLKEHKALKTLGIIMGTFTLCWLPFFIVN
ClassA_stamp_VMDA2A --------------CH---HHHH-----------------------------------------------------------H------HH-HHHCHHHHHHHHHHHHHHHHHHHHHHHH ClassA_stamp_VMDAvi ---------------------------------------------------------------------------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHH ClassA_stamp_VMDBov --------------------------------------------------------------------------------T--TCCCCCCH-HHHHHHHHHHHHHHHHHHHHHHHHHHHH ClassA_stamp_VMDHum TTHHHHHHHCCHHHHHHHHH---HHHHHHHHHHHHCHHHHHHHHHCCHHHHHHHHHCCHHHHHCHHHHHHHHHHHHHCCCGGGTT------TCHHHHHHHHHHHHHHHHHHHHHHHHHHH
ClassA_stamp_VMDA2A CFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLR--Q---------------- ClassA_stamp_VMDAvi IVNVFNRDL--VPDWLFVAFNWLGYANSAMNPIIYC-RSPDFRKAFKRLLA------------------------ ClassA_stamp_VMDBov FYIFTHQGSD-FGPIFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCC----GKNPSTTVSKTETSQVAPA ClassA_stamp_VMDHum IVHVIQDNL--IRKEVYILLNWIGYVNSGFNPLIYC-RSPDFRIAFQELLC-------L----------------
ClassA_stamp_VMDA2A HHHHHTTTTCCCCHHHHHHHHHHHHHHCHHHHHHHHHHCHHHHHHHHHHHHHHHCC--C---------------- ClassA_stamp_VMDAvi HHHHHTTTT--CCHHHHHHHHHHHHHHHHHHHHHHH-HCHHHHHHHHHHHC------------------------ ClassA_stamp_VMDBov HHHHHTTTTC-CCHHHHHHHHHHHGGGGHHHHHHHHHHCHHHHHHHHHHHHT----TTCCCCCCTTTTTTCCCCC ClassA_stamp_VMDHum HHHHHTTTT--TTHHHHHHHHHHHHHHHCHHHHHHH-CCHHHHHHHHHHHC-------C----------------
Fig 21. Structural alignment of class A receptors
3. GPCRsStructural Conservation
SCORE 5.57RMSD 1.92
Rhodopsin
β2-Adrenergic Receptor
A2-Adenosin Receptor
β1-Adrenergic Receptor
Fig22. Superimposition of β2AR, β1AR, A2AR and Rhodopsin Fig23. Superimposition of β2AR, β1AR, A2AR and
Rhodopsin
β2 Adrenergic Receptor
Fig25. β2 Adrenergic Receptor sequence
>beta 2 adrenergic receptor [Homo sapiens] MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMAFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL
β2AR
Adenilate
cyclase
G proteins
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
Fig27. Hydropathy plot of β2 Adrenergic Receptor sequence
3. GPCRs
Fig26 Hydropathy plot of β2 Adrenergic Receptor sequence
MEMSAT software to predict alpha helix transmembrane regions based on Dr. Jones predictive algorithm described in 1994
β2 Adrenergic Receptor
Fig28. β2 Adrenergic Receptor
ECL2
III
I
II
VIII
VI
V
VIIIV
carazolol
T4-lysozyme
Extracellular
Intracellular
3. GPCRs
β2 Adrenergic Receptor TM1
TM2TM3 TM4
TM5
TM6TM7
Fig29. Sequence and secondary structure of β2 Adrenergic Receptor sequence obtained from PDB
3. GPCRs
Fig30. β2 Adrenergic Receptor
ECL2
III
I
II
VIII
VI
V
VIIIV
carazolol
T4-lysozyme
Extracellular
Intracellular
β2 Adrenergic Receptor
3. GPCRs
ECL2 (Extracellular Loop 2)
ECL2
Carazolol
Phe 193
Cys 106
Cys 190
Cys 191
Cys 184
Fig31. Extracellular Loop 2 of the β2 Adrenergic Receptor
3. GPCRs
ECL2 (Extracellular Loop 2)
ECL2
Carazolol
Phe 193
Cys 106
Cys 190
Cys 191
Cys 184
β2-Adrenergic Receptor
Rhodopsin
ECL2
Retinal
Cys 110
Cys 187
Fig32. Extracellular Loop 2 of the β2 Adrenergic Receptor
Fig33. Extracellular Loop 2 of Rhodopsin
3. GPCRs
ECL2 (Extracellular Loop 2)
Rhodopsin
β2-Adrenergic Receptor
A2-Adenosin Receptor
Fig34. Extracellular Loop 2 of β2-Adrenergic Receptor, Rhodopsin and A2- Adenosin Receptor with the ligands carazolol and retinal
Fig35. Extracellular Loop 2 of β2-Adrenergic Receptor, Rhodopsin and A2- Adrenergic Receptor with their conserved disulfur bonds.
Fig36. Extracellular Loop 2 of A2- Adenosin Receptor with its three disulfur bonds.
ECL2
ECL1
Cys 74
Cys 146
Cys 71
Cys 159
Cys 77
Cys 166
3. GPCRs
β2 Adrenergic Receptor
Fig37. β2 Adrenergic Receptor
ECL2
III
I
II
VIII
VI
V
VIIIV
carazolol
T4-lysozyme
Extracellular
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
Fig39. Interaction between β2-Adrenergic Receptor and carazolol
Fig40. Interaction between β2-Adrenergic Receptor and carazolol
Ser203
Ser203
Ser203
Carazolol
Asn312
Asp113
Ser203
Asn312
Asp113
Carazolol
3. GPCRs
Ligand binding site
Fig41. Interaction between β2-Adrenergic Receptor and carazololFig42. Interaction between β2-Adrenergic Receptor and carazolol
Trp109
Phe290
Tyr199
Phe289
Phe193
Trp286
Val114
Trp109
Phe193
Tyr199
Val114
Phe290 Phe2
89
Trp286
3. GPCRs
Fig43. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin and retinal
Ligand binding site
3. GPCRs
Ligand binding site
3. GPCRs
Fig44. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin and retinal
Ligand binding site
3. GPCRs
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
Phe5.47
Tyr6.51
Trp6.48
Tyr6.51
Trp6.48
Phe5.47
Ligand binding site
3. GPCRs
Fig47. Interaction between Rhodopsin and retinal through Trp6.48
Fig48. Interaction between β2-Adrenergic Receptor and carazolol and Trp6.48
Trp6.48
Trp6.48
β2-Adrenergic Receptor
Rhodopsin
“Toogle Switch”
β2 Adrenergic Receptor
Fig49. β2 Adrenergic Receptor
ECL2
III
I
II
VIII
VI
V
VIIIV
carazolol
T4-lysozyme
Extracellular
Intracellular
3. GPCRs
Ionic Lock
3. GPCRs
Fig50. Ionic lock of Rhodopsin Fig51. Ionic lock of β2-Adrenergic Receptor
β2-Adrenergic Receptor
Rhodopsin
Conserved E/D(E)RY motif
Fig52. Superimposition of the Ionic lock of Rhodopsin and β2-Adrenergic Receptor
Tyr3.51
Glu3.49
Glu6.30
Arg3.50
Tyr3.51
Asp3.49
Glu6.30
Arg3.50
Tyr3.51
Glu/Asp3.49
Glu6.30
Arg3.50
Activation
3. GPCRs
Fig53. β2 Adrenergic Receptor
ECL2
III
I
II
VIII
VI
V
VIIIV
carazolol
T4-lysozyme
Extracellular
Intracellular
Fig54. Nanobody stabilized-β2 Adrenergic Receptor
ECL2
II
I
VIII
VI
VII
BI-167107
Nanobody (Nb80)
Extracellular
Intracellular
V
III
IV
Activation
3. GPCRs
ECL2
III
I
II
VI
V
VII
IV
ICL2
III
I
II
VI
V
VII
IV
Fig56. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
Fig57. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
Inactive state
Active state
Activation
3. GPCRs
Trp109
Phe193Tyr19
9
Val114
Phe290
Phe289
Trp286
Tyr199
Phe290
Trp286
Phe289
Ile309
Phe193
Val114
Fig58. Interaction between β2-Adrenergic Receptor and BI-167107
Fig59. Interaction between β2-Adrenergic Receptor and carazolol
Activation
3. GPCRs
Fig61. Interaction between β2-Adrenergic Receptor and carazolol
Ser203
Asn312
Asp113
Carazolol
Ser203
Asn312
Asp113
BI-167107
Fig60. Interaction between β2-Adrenergic Receptor and BI-167107
Fig62. Interaction between β2-Adrenergic Receptor and BI-167107
Ser203
Ser207
Asn312
BI-167107
Activation
3. GPCRs
carazolol
Ser203Ser207
Pro211
Ile121
Phe282
Asn318
Ser203
Ser207
BI-167107
Fig63. β2 Adrenergic Receptor in its inactivated state. Fig64. Adrenergic Receptor in its activated state.
Fig65. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
Pro2115.50
Ile1213.40
Phe2826
.44
Asn3187
.45
Phe2826
.44
Ser2075.46
Pro2115.50
Ile1213.40
Phe2826
.44
Asn3187
.45
Activation
3. GPCRs
Fig66. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
Trp6.48
Activation
3. GPCRs
Fig67. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states.
Glu268
Arg131
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 β2- Adrenergic 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: 6843–6848-
López 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. 3-39. 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-Protein-Coupled 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.