MPNNSTALSLANVTYITMEIFIGLCAIVGNVLVICVVKLNPSLQTTTFYFIV

SLALADIAVGVLVMPLAIVVSLGITIHFYSCLFMTCLLLIFTHASIMSLLAIA

VDRYLRVKLTVRYKRVTTHRRIWLALGLCWLVSFLVGLTPMFGWNMKL

TSEYHRNVTFLSCQFVSVMRMDYMVYFSFLTWIFIPLVVMCAIYLDIFYII

RNKLSLNLSNSKETGAFYGREFKTAKSLFLVLFLFALSWLPLSIINCIIYFN

GEVPQLVLYMGILLSHANSMMNPIVYAYKIKKFKETYLLILKACVVCHPS

DSLDTSIEKNSEPNNSTALSLANVTYITMEIFIGLCAIVGNVLVICVVKLN

PSLQTTTFYFIVSLALADIAVGVLVMPLAIVVSLGITIHFYSCLFMTCLLLI

FTHASIMSLLAIAVDRYLRVKLTVRYKRVTTHRRIWLALGLCWLVSFLVG

LTPMFGWNMKLTSEYHRNVTFLSCQFVSVMRMDYMVYFSFLTWIFIPL

VVMCAIYLDIFYIIRNKLSLNLSNSKETGAFYGREFKTAKSLFLVLFLFAL

SWLPLSIINCIIYFNGEVPQLVLYMGILLSHANSMMNPIVYAYKIKKFKET

YLLILKAC VVCHPSDSLDTSIEKNSE

Tertiary Structure Prediction of Adenosine A3 Receptor

Presented by Lindsay RileyGoddard Group, Caltech

SoCalBSI, CalStateLA20 August 2009

Significance of Adenosine A3 Receptor

Good target for Important to know structure for drug specification

BACKGROUND• G-protein-coupled receptor (GPCR)

7 transmembrane helices exterior interior

Involved in many cell signaling pathways drug therapy

http://www.biomedcentral.com/content/figures/1471-2105-8-177-1-l.jpg

Significance of Adenosine A3 Receptor

FUNCTION• Prevents restriction of blood flow in brain and

heart• Anti-inflammatory, anti-cancer effects• Protection of spinal cord and bone marrow

Kim, S. , Jacobson, K. “Three-Dimensional Quantitative Structure – Activity Relationship of Nucleosides Acting at the A3 Adenosine Receptor: Analysis of Binding and Relative Efficacy.” J. Chem. Inf. Model. 2007, 47, 1225-1233

THERAPEUTIC IMPLICATIONS• A3 ligand (IB-MECA) in Phase II clinical trials for treatment

of rheumatoid arthritis and metastatic colorectal tumors

HINDRANCES• Difficult to perform X-ray

crystallography on transmembrane proteins

• Only 2 / 800 human GPCR structures determined via X-ray crystallography:

• Bovine rhodopsin• Squid rhodopsin• Turkey β1-adrenergic receptor• Human β2-adrenergic receptor• Human A2a-adenosine receptor

SOLUTION• Predict structure using all-atom based

first principles methods

GEnSeMBLEHomology Helix1. Align and homologize2. Optimize helices3. BiHelix4. CombiHelix5. Analyze top

structures6. SuperBiHelix

Final Structure

1. Hydrophobicity profile2. OptHelix3. Align to template4. BiHelix5. Combihelix6. Analyze top structures7. SuperBiHelix

Final Structure

MPNNSTALSLANVTYITMEIFIGLCAIVGNVLVICVVKLNPSLQ

TTTFYFIVSLALADIAVGVLVMPLAIVVSLGITIHFYSCLFMTCLL

LIFTHASIMSLLAIAVDRYLRVKLTVRYKRVTTHRRIWLALGLC

WLVSFLVGLTPMFGWNMKLTSEYHRNVTFLSCQFVSVMRMD

YMVYFSFLTWIFIPLVVMCAIYLDIFYIIRNKLSLNLSNSKETGAF

YGREFKTAKSLFLVLFLFALSWLPLSIINCIIYFNGEVPQLVLYMG

ILLSHANSMMNPIVYAYKIKKFKETYLLILKAC

VVCHPSDSLDTSIEKNSE

GEnSeMBLE

1. Align and homologize

2. Optimize helices3. BiHelix4. CombiHelix5. Analyze top structures6. SuperBiHelix

Homology Helix

CLUSTAL 2.0.11 multiple sequence alignment

sp|P33765|AA3R_HUMAN MPNNSTALSLANVTYITMEIFIGLCAIVGNVLVICVVKLNPSLQTTTFYF 50sp|P29274|AA2AR_HUMAN MP------IMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYF 44 ** :.. .***:*: *.: **:***** .* **..**..* **

sp|P33765|AA3R_HUMAN IVSLALADIAVGVLVMPLAIVVSLGITIHFYSCLFMTCLLLIFTHASIMS 100sp|P29274|AA2AR_HUMAN VVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFS 94 :**** ********.:*:**.:* *: :.***::*::*::*::**:*

sp|P33765|AA3R_HUMAN LLAIAVDRYLRVKLTVRYKRVTTHRRIWLALGLCWLVSFLVGLTPMFGWN 150sp|P29274|AA2AR_HUMAN LLAIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWN 144 *****:***: :::.:**: :.* * :.:**::** :*****:***

sp|P33765|AA3R_HUMAN ---MKLTSEYHRN---VTFLSCQFVSVMRMDYMVYFSFLTWIFIPLVVMC 194sp|P29274|AA2AR_HUMAN NCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLML 194 .: * : ::* * .*: *:*****.*:: :::**::*

sp|P33765|AA3R_HUMAN AIYLDIFYIIRNKLSLNLSN---SKETGAFYGREFKTAKSLFLVLFLFAL 241sp|P29274|AA2AR_HUMAN GVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFAL 244 .:** ** *.:*. *: .:.: : :*.::**** ::: ****

sp|P33765|AA3R_HUMAN SWLPLSIINCIIYFN---GEVPQLVLYMGILLSHANSMMNPIVYAYKIKK 288sp|P29274|AA2AR_HUMAN CWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIRE 294 .**** ****: :* ...* ::*:.*:***:**::**::***:*::

sp|P33765|AA3R_HUMAN FKETYLLILKACVV-----------------CHPSDSLDTSIEKNSE--- 318sp|P29274|AA2AR_HUMAN FRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPG 344 *::*: *::: *: .* **. :.*:. *..

sp|P33765|AA3R_HUMAN --------------------------------------------------sp|P29274|AA2AR_HUMAN VWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVC 394

sp|P33765|AA3R_HUMAN ------------------sp|P29274|AA2AR_HUMAN PEPPGLDDPLAQDGAGVS 412

Alignment of A3 sequence to A2a template

Side View Top View

MPNNSTALSLANVTYITMEIFIGLCAIVGNVLVICVVKLNPSLQ

TTTFYFIVSLALADIAVGVLVMPLAIVVSLGITIHFYSCLFMTCLL

LIFTHASIMSLLAIAVDRYLRVKLTVRYKRVTTHRRIWLALGLC

WLVSFLVGLTPMFGWNMKLTSEYHRNVTFLSCQFVSVMRMD

YMVYFSFLTWIFIPLVVMCAIYLDIFYIIRNKLSLNLSNSKETGAF

YGREFKTAKSLFLVLFLFALSWLPLSIINCIIYFNGEVPQLVLYMG

ILLSHANSMMNPIVYAYKIKKFKETYLLILKAC

VVCHPSDSLDTSIEKNSE

GEnSeMBLE

1. Hydrophobicity profile

2. OptHelix3. Align to template4. BiHelix5. CombiHelix6. Analyze top

structures7. SuperBiHelix

GEnSeMBLE

GEnSeMBLE

Homology Helix

Secondary Structure Prediction

70 80 90 100 110 120 | | | | | |

SEQ: VGVLVMPLAIVVSLGITIHFYSCLFMTCLLLIFTHASIMSLLAIAVDRYLRVKLTVRYKR PORTER: HHHccHHHHHHHHHHcccccHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHccccccccPSIPRED: HHHHHHHHHHHHHccccccccHHHHHHHHHHHHHHHHHHHHHHHHHHHEEEEcccccccEOLD_RAW: HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------------OLD_CAP: HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------------

190 200 210 220 230 240 | | | | | | SEQ: SFLTWIFIPLVVMCAIYLDIFYIIRNKLSLNLSNSKETGAFYGREFKTAKSLFLVLFLFA PORTER: HHHHHcHHHHHHHHHHHHHHHHHHHHHHccccccccccccHHHHHHHHHHHHHHHHHHHHPSIPRED: HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHcccccHHHHHHHHHHHHHHHHHHHHHHOLD_RAW: HHHHHHHHHHHHHHHHHHHH--------------------------------HHHHHHHHOLD_CAP: HHHHHHHHHHHHHHHHHHHHHHHHH------------------------HHHHHHHHHHH

TM2 & TM3

TM6

OptHelix

* Proline and Glycine residues displayed

Alignment of A3 helices to A2a template

GEnSeMBLEHomology Helix1. Align and homologize2. Optimize helices3. BiHelix4. CombiHelix5. Analyze top

structures6. SuperBiHelix

Final Structure

1. Hydrophobicity profile2. OptHelix3. Align to template4. BiHelix5. Combihelix6. Analyze top structures7. SuperBiHelix

Final Structure

BiHelix: energy minimization of helix pairs

CombiHelix Output

Homology HelixH1 H2 H3 H4 H5 H6 H7 Energy0 0 0 15 0 0 0 2930 0 0 0 0 0 0 3000 0 15 15 0 15 0 3100 0 0 0 0 30 345 3120 0 0 15 0 15 0 3150 0 15 345 0 15 0 3240 0 0 15 0 30 0 3250 0 0 0 0 15 0 3250 0 0 330 0 15 0 3380 0 0 345 0 30 345 339

Dock Ligands

DarwinDock Matching

GEnSeMBLEHomology Helix

Final Structure Final Structure

or

Dock Ligands

DarwinDock Matching

GEnSeMBLEHomology Helix

Final Structure Final Structure

or

DarwinDock: adenosine

adenosine

Important residues involved in agonist binding (based on mutation experiments)

6 ASP

7 HIS3 HIS

7 SER

Matching: adenosine

Also working on…• analyzing A3 adenosine docking• analyzing A3 SuperBiHelix output• A2a structure and ligand docking• A1 structure and ligand docking• A2b structure and ligand docking• docking agonists: ClIBMECA & NECA• docking antagonists: MRS1292 &

NMeClIBMECA

To do list…• add loops to final structure• simulate lipid bilayer

Acknowledgements

Funding

Goddard Group at California Institute of Technology

Dr. Soo-Kyung KimDr. Ravinder AbrolProfessor William A. Goddard IIIBiogroup

SoCalBSI at California State University, Los Angeles

Dr. Jamil MomandDr. Sandy SharpDr. Nancy Warter-PerezDr. Wendie JohnstonRonnie ChengThe lovely interns

NIH, Pfizer, PharmSelex