Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS.

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Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS

Transcript of Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS.

Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso

MEMBRANEPROTEINS

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

3. GPCRsNomenclature

Fig 20. Scheme of structure of the class A

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

3. GPCRsStructural Conservation

Fig24. 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

Fig55. Conserved prolines in β2 Adrenergic Receptor

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

Activation

3. GPCRs

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.

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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.

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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.

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