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

2. 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.Conclusions 5.Bibliography

3. 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:

4. Extracellular region Intracellular region Transmembrane region Transmembrane proteins DomainFunctions Extracellular Involved in cell – cell signaling Let interactions with other molecules TransmembraneForms channels and pores IntracellularActivates intracellular signaling pathways 1. Introduction Fig3. Cell membrane with a transmembrane protein.

5. 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

6. 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

7. 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.Conclusions 5.Bibliography

8. 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 proteinFig5. OmpA protein Extracellular Intracellular Fig6. NalP proteinFig7. OmpA protein Extracellular Intracellular

9. 2. Types of transmembrane proteins α - Helical Extracellular Fig9. Rodhopsin Intracellular Fig8. PufX protein It contains a hydrophilic domain on each side of the membrane Bitopic It contains a multiple hydrophilic domains on both sides of the membrane Polytopic Fig10. Bitopic proteins and Polytopic proteins

10. 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.Conclusions 5.Bibliography

11. 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 GPCR The most important family of membrane proteins

12. 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

13. 3. GPCRs Sequence 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

14. 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

15. Class A. SCOP Fig 18. Class A classification from SCOP (Structural Classification of Proteins) 3. GPCRs

16. Class A. Chronology Before 20002000200720082008-2012 Rhodopsin Palczewski et al. Aug. 2000 β1 -Adrenergic Receptor Warne et al. July 2008 2-Adenosine Receptor Jaakola et al. Oct. 2008 β2-Adrenergic Receptor D1-R H1-R SPS1 … 3. GPCRs Cherezov et al. Nov. 2007

17. TM7 TM6 TM5 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-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-hydroxytrypIFCAVLGNACVVAAIAL..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-P beta-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.IKQG D2 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-G histamine TS---.---KGNHTTS-----KCKVQ--V--------NEVY-GLVDG...LVTFYLPLLIMCITYYR....I...FKVAR...KAT....V...TLAAVMGAFIICWFPYFT...AF....VYR...G.LR-G melatonin -----.---EYDPRIY-----SCT-F--IQ-----TASTQY-TAAVV...VIHFLLPIAVVSFCYLR....IwvlVLQAR...RSF....L...TMFVVFVIFAICWAPLNC...IG....LAV...A.IN-P muscarinic QF--I.VGVRTVEDGE------CYIQ--FF------SNAAV-TFGTA...IAAFYLPVIIMTVLYWH....I...SRASK...VAN....QdpvSPSLVQG---------RI...VK....PNN...N.NM-P delta 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.LGST Mu-type ----T.K--YRQGSID------CTLT--FS--HPTWYWENLLKICVF...IFAFIMPVLIITVCYGL....M...ILRLK...RIT....R...MVLVVVAVFIVCWTPIHI..yVI....IKA...L.VTIP orexin ECSSVlP--ELANRTR--LFSVCD--ERWA---DDLYPKIY-HSCFF...IVTYLAPLGLMAMAYFQ....I...FRKLW...KTA....K...MLMVVLLVFALCYLPISV...LNvlkrVFG...M.FRQA rhodopsin -----.---R---YIPEGLQCSCGID--YYTLKPEVNNESF-VIYMF...VVHFTIPMIIIFFCYGQ....L...VFTVK...EVT....R...MVIIMVIAFLICWVPYAS...VA....FYI...F.THQG 5-hydroxytrypED---.---R-SDPDA------CTISKDH----------GY-TIYST...FGAFYIPLLLMLVLYGR....I...FRAARfriKTV....K...TLGIIMGTFILCWLPFFI...VA....LVL...P.FCES somatostatin EGGTC.NASW---PEP------VGLW-----------GAVF-IIYTA...VLGFFAPLLVICLCYLL....I...VVKVR...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 3. GPCRs Fig 19.Sequence aligment of many class A receptors based on hmm model

18. 3. GPCRs Nomenclature Fig 20. Scheme of structure of the class A

19. Lysozyme TM1TM2 TM3TM4 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

20. 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

21. 3. GPCRs Structural Conservation Fig24. Superimposition of β2AR, β1AR, A2AR and Rhodopsin

22. β2 Adrenergic Receptor Fig25. β2 Adrenergic Receptor sequence >beta 2 adrenergic receptor [Homo sapiens] MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKF ERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIET LCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYA NETCCDFFTNQAYAIASSIVSFYVPLVIMAFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVE QDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNW IGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKL LCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL 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

23. 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

24. β2 Adrenergic Receptor Fig28. β2 Adrenergic Receptor ECL2 III I II VIII VI V VIIIV carazolol T4-lysozyme Extracellular Intracellular 3. GPCRs

25. β2 Adrenergic Receptor TM1 TM2 TM3TM4 TM5 TM6 TM7 Fig29. Sequence and secondary structure of β2 Adrenergic Receptor sequence obtained from PDB 3. GPCRs

26. Fig30. β2 Adrenergic Receptor ECL2 III I II VIII VI V VIIIV carazolol T4-lysozyme Extracellular Intracellular β2 Adrenergic Receptor 3. GPCRs

27. 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

28. ECL2 (Extracellular Loop 2) ECL2 Carazolol Phe 193Cys 106 Cys 190 Cys 191 Cys 184 ECL2 Retinal Cys 110 Cys 187 Fig32. Extracellular Loop 2 of the β2 Adrenergic Receptor Fig33. Extracellular Loop 2 of Rhodopsin 3. GPCRs

29. ECL2 (Extracellular Loop 2) 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

30. β2 Adrenergic Receptor Fig37. β2 Adrenergic Receptor ECL2 III I II VIII VI V VIIIV carazolol T4-lysozyme Extracellular Intracellular 3. GPCRs

31. Ligand binding site Fig38. Extracellular view of the ligand binding site of the β2 Adrenergic Receptor with its ligand carazolol 3. GPCRs

32. Ligand binding site Fig39. Interaction between β2-Adrenergic Receptor and carazolol Fig40. Interaction between β2-Adrenergic Receptor and carazolol Ser203 Carazolol Asn312 Asp113 Ser203 Asn312 Asp113 Carazolol 3. GPCRs

33. 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 Phe289 Trp286 3. GPCRs

34. Fig43. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin and retinal Ligand binding site 3. GPCRs

35. Ligand binding site 3. GPCRs Fig44. Interaction between β2-Adrenergic Receptor and carazolol compared to the interaction between Rhodopsin and retinal

36. 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

37. Ligand binding site 3. GPCRs Fig47. Interaction between Rhodopsin and retinal through Trp6.48Fig48. Interaction between β2-Adrenergic Receptor and carazolol and Trp6.48 Trp6.48 “Toogle Switch”

38. β2 Adrenergic Receptor Fig49. β2 Adrenergic Receptor ECL2 III I II VIII VI V VIIIV carazolol T4-lysozyme Extracellular Intracellular 3. GPCRs

39. Ionic Lock 3. GPCRs Fig50. Ionic lock of Rhodopsin 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 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

40. 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

41. Activation 3. GPCRs Fig55. Conserved prolines in β2 Adrenergic Receptor

42. Activation 3. GPCRs ECL2 III I II VIV 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.

43. Activation 3. GPCRs Trp109Phe193 Tyr199 Val114 Phe290 Phe289 Trp286 Tyr199 Phe290 Trp286Phe289 Ile309 Phe193 Val114 Fig58. Interaction between β2-Adrenergic Receptor and BI-167107Fig59. Interaction between β2-Adrenergic Receptor and carazolol

44. 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

45. Activation 3. GPCRs carazolol Ser203 Ser207 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. Pro211 5.50 Ile121 3.40 Phe282 6.44 Asn318 7.45 Phe282 6.44 Ser207 5.46 Pro211 5.50 Ile121 3.40 Phe282 6.44 Asn318 7.45

46. Activation 3. GPCRs Fig66. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states. Trp6.48

47. Activation 3. GPCRs Fig67. Superimposition of β2 Adrenergic Receptor in its activated and inactivated states. Glu268 Arg131

48. Activation 3. GPCRs Glu268 Arg131 Fig68. β2 Adrenergic Receptor coupled to G-protein

49. 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.

50. 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 β 2 - 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 β 2 - 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 β 2 - 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 β 2 -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.

51. 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 β 2 - 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 β 2 - 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.

52. 5. Bibliography Rasmussen S.G, et al. Crystal structure of the human β 2 -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 β 2 Adrenergic Receptor-Gs protein complex. Nature. 2011; 477: 549-555. Rasmussen S.G, et al. Structure of a nanobody-stabilized active state of the β 2 - adrenoceptor. Nature. 2011; 469: 175–180. Rosenbaum D.M,et al. GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function. Science. 2007; 318: 1266–1273 Rosenbaum D. M, Rasmussen S. G, Kobilka B.K. The β 2 - 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 β 2 - 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

53. 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 β 1 - 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.

54. THANKS FOR YOUR ATTENTION!!!! QUESTIONS?