STRUCTURE FUNCTION STUDIES OF THE ALPHA PHEROMONE RECEPTOR FROM YEAST STRUCTURE FUNCTION STUDIES OF THE ALPHA PHEROMONE RECEPTOR FROM YEAST Laura Marina Robles1, César Millán Pacheco3, Nina Pastor2 an[.]
Trang 1S TRUCTURE - FUNCTION STUDIES OF THE ALPHA
Laura Marina Robles 1 , César Millán-Pacheco 3 ,
Nina Pastor 2 and Gabriel Del Río 1 *
1 Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Deleg Coyoacán, C.P 04510, Ciudad de México, México 2 Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos, Av Universidad #1001, Col Chamilpa, Cuernavaca , Morelos, C.P 62209, México 3 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av Universidad #1001, Col Chamilpa,
Cuernavaca , Morelos, C.P 62209, México E-mail: *gdelrio@ifc.unam.mx
Nota: Artículo recibido el 28 de julio de 2016 y aceptado el
03 de noviembre de 2016
ABSTRACT
Ste2p is a G protein-coupled receptor (GPCR) in Saccharomyces cerevisiae that mediates mating by responding
to the alpha-mating factor pheromone Ste2p belongs to a subfamily of GPCRs with no global sequence similarity
to GPCRs of known atomic three-dimensional structure, yet it shares functional similarities with many of these
To deepen our understanding of the structure-function relationship of this receptor, we built an atomic three-dimensional homology-based model of Ste2p that was used to simulate the docking of the alpha pheromone The Ste2p model is in general agreement with the available experimental data and allowed us to propose that the interface between Ste2p and alpha pheromone is formed by 26 residues, most of which are polar residues located at the three extracellular loops and helices HI, H5, and H6 This interface does not include Ile190, a highly conserved residue among fungal species, located at the second extracellular loop and therefore a potential
binding site residue By performing mutagenesis of STE2 at this position we observed only a small effect of this residue
in receptor signaling Hence, the Ste2p model presented here is consistent in general with current experimental data and constitutes a framework to test hypothesis about the structure-function relationship of this receptor.
Key Words: alpha pheromone receptor, docking, molecular modeling, pheromone, Ste2p.
Estudio de la relación entre la estructura y la función del receptor de la feromona alfa de levadura
RESUMEN
Ste2p es un receptor acoplado a la proteína G (GPCR) en Saccharomyces cerevisiae que se une a la feromona
alfa para mediar el apareamiento Ste2p pertenece a una subfamilia de GPCRs que no presentan homología global en secuencia con los GPCRs de estructura atómica tridimensional conocida, pero comparte propiedades funcionales con muchos de éstos Para profundizar nuestro entendimiento de la relación estructura-función
de este receptor, en este trabajo presentamos un modelo de la estructura atómica tridimensional de Ste2p asociado a su ligando El modelo de Ste2p generado es congruente con la información experimental disponible
y sugiere que la interfaz entre Ste2p y la feromona está compuesta por 26 residuos, en su mayor parte polares, localizados en las tres asas extracelulares y las hélices H1, H5 y H6 La interfaz no incluye a la Ile190, un residuo altamente conservado entre especies de hongos, que se encuentra en el asa extracelular 2 y es un potencial
sitio de anclaje Mutantes en esta posición en STE2 muestran un efecto pequeño en la señalización del receptor
El modelo presentado de Ste2p es consistente en general con los datos de mutagénesis disponibles a la fecha, por lo que constituye un marco de referencia para evaluar hipótesis sobre la relación estructura-función en este receptor.
Palabras Clave: receptor de la feromona alfa, anclado molecular simulado, modelado molecular, feromona, Ste2p
This is an Open Access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0).
TIP Revista Especializada en Ciencias Químico-Biológicas, 20(1): 16-26, 2017.
DOI: 10.1016/j.recqb.2016.11.002
Trang 2ver the past four decades extensive research has been
carried out about the structure-function relationships
of G protein coupled receptors (GPCRs), promoted
by both basic and applied aspects of this group of
INTRODUCTION
O
receptors; more than 40% of drugs in clinical use target GPCRs1
and many fundamental aspects of cell physiology are regulated
by these receptors2 Relevant to this study, the GPCR Ste2p from
Saccharomyces cerevisiae mediates mating by recognizing the
tridecapeptide mating factor, alpha pheromone
7KH¿UVWVWXGLHVRQ6WHSZHUHUHSRUWHGLQ ZKHQWKH
PROHFXODUEDVLVRIPDWLQJLQ\HDVWFHOOVZDVGLVFRYHUHG0DQ\
JHQHVUHQGHULQJDVWHULOH 67( SKHQRW\SHZHUHGLVFRYHUHG
DQG DPRQJ WKHVH ZDV STE23,4 WKDW ZDV IXUWKHU FORQHG DQG
characterized5,6$ IHZ \HDUV ODWHU WKH WRSRORJLFDO VWUXFWXUH
RI 6WHS ZDV LQIHUUHG WKURXJK VROYHQW DFFHVVLELOLW\ VWXGLHV
)LJXUH 'XHWRGL൶FXOWLHVWRSXULI\DQGFU\VWDOOL]HLQWHJUDO
membrane proteins, the atomic three-dimensional structure of
6WHSKDVQRW\HWEHHQGHWHUPLQHG+RZHYHU105ZDVXVHG
WRGHWHUPLQHWKHVWUXFWXUHVRIWZRIUDJPHQWVRIWKLVUHFHSWRU
WKH+,+VHJPHQW 3URWHLQ'DWD%DVHHQWU\FRGH . 3 DQG
DVHJPHQWRI+ 3'%HQWU\FRGH 3-'
Several residues and regions that are critical for STE2 function
have been determined by site-directed mutagenesis For
H[DPSOHWKH1WHUPLQDOGRPDLQPHGLDWHVROLJRPHUL]DWLRQRI
the receptor ZKLOHWKH&WHUPLQDOGRPDLQDFWVDVQHJDWLYH
regulator11-13 7KH WKLUG F\WRSODVPLF ORRS LQWHUDFWV ZLWK WKH
trimeric G protein14DQGWKH¿UVWH[WUDFHOOXODUORRSXQGHUJRHV
a conformational change upon ligand binding15 and also plays
a role in signal transduction16
7KUHHGL൵HUHQW'PRGHOVRI6WHSERXQGWRWKHSKHURPRQHKDYH EHHQSURSRVHGDQGZHUHEXLOWEDVHGRQELRFKHPLFDOGDWDGHULYHG until 2004 in an attempt to recapitulate the structure-function VWXGLHV RI WKLV UHFHSWRU %ULHÀ\ /HH % DQG FROOHDJXHV SURSRVHG D PRGHO ZKHUH ERWK WKH 1 DQG &WHUPLQL RI WKH pheromone are buried inside the transmembrane helices of Ste2p, ZKHUHDVWKHFHQWUDOUHJLRQLQFOXGLQJDWXUQVWUXFWXUHLQWHUDFWV ZLWKWKHH[WUDFHOOXODUGRPDLQV,QWKDWPRGHOWZRUHVLGXHVRI WKHUHFHSWRU6HUDQG7KUZHUHSURSRVHGWRLQWHUDFWZLWK
*OQRIWKHSKHURPRQH/LQ-&DQGFROOHDJXHV proposed
a model that highlights the role of aromatic residues in the LQWHUDFWLRQEHWZHHQWKHSKHURPRQHDQGWKHSRFNHWIRUPHGE\WKH extracellular ends of the transmembrane helices in the receptor ,QWKLVPRGHODȕEHQGLVIRUPHGDWWKHFHQWUDOUHJLRQRIWKH SKHURPRQH7\URQKHOL[LVRULHQWHGWRZDUGWKHVXUURXQGLQJ OLSLGV DQG LQWHUDFWV ZLWK7US+LV7US RI WKH SKHURPRQH ZKLOH3KHORFDWHGZLWKLQWKHKHOL[EXQGOHEHWZHHQKHOL[
DQGKHOL[LQWHUDFWVZLWK7\URIWKHSKHURPRQH)LQDOO\ Son C D and colleagues SURSRVHGDPRGHOZKHUH*OQRI WKHSKHURPRQHLQWHUDFWVZLWKUHVLGXHV6HUDQG7KULQ6WH 7\ULQWKHSKHURPRQHLQWHUDFWVZLWKKHOL[ 3KHí$UJ DQG7USDQG7USLQWKHSKHURPRQHLQWHUDFWZLWKDURPDWLF residues Phe262 and Tyr266 at the extracellular interface of helix 6 All these models have in common that the central region of the pheromone forms a turn structure that is oriented DZD\ IURP WKH WUDQVPHPEUDQDO KHOL[EXQGOH ZKHUHDV ERWK WKH1DQG&WHUPLQLRIWKHSKHURPRQHDUHRULHQWHGWRZDUGWKH binding pocket formed by extracellular ends of helix 1, helix
5, and helix 6
,QWKHSUHVHQWVWXG\ZHEXLOWDQRYHOWKUHHGLPHQVLRQDOPRGHO
of Ste2 bound to the pheromone, incorporating mutagenesis
Figure 1 Topological diagram of Ste2p Every amino acid residue from Ste2p is represented by a single letter code within a gray circle The two horizontal lines separating the extracellular from the intracellular spaces represent the membrane Data derived from 20
Trang 3/HX/\V3UR*O\*OQ3UR0HW7\UZDVVHQWWRWKHVHUYHULQ )$67$ IRUPDW 7KH PRGHO WKDW ZDV FORVHU WR WKH VWUXFWXUDO FKDUDFWHUL]DWLRQRIWKHSKHURPRQHE\10531ZDVVHOHFWHGIURP WKHUHVXOWVJHQHUDWHGE\WKH3(3)2/'VHUYHU
Docking of Ste2 with alpha pheromone For docking experiments
the ClusPro 2.0 server32ZDVXVHG$PLQRDFLGUHVLGXHVRIWKH 6WHSUHFHSWRUDQGWKHSKHURPRQHLQYROYHGLQELQGLQJZHUHXVHG
to focus the docking on such residues These residues included (Ste2p-pheromone pairs; the secondary structure element of WKH6WHSUHVLGXHLVLQGLFDWHGLQSDUHQWKHVLVIRUFRQYHQLHQFH 6HU + *OQ7KU + *OQ, Phe204(H5)-Tyr13, Asn205(H5)-Trp333, Tyr266(H6)-Trp3 DQG /\V + Trp134 The energy of the Ste2p-alpha pheromone complex ZDV PLQLPL]HG XVLQJ &+$50035 IRU WKDW HQG WKH &%
&+$500 YHUVLRQ DQG &+$500 SRWHQWLDOV ZHUH XVHG 7KHFRPSOH[WKDWVKRZHGWKHORZHVWLQWHUDFWLRQHQHUJ\ZLWK WKHORZHVWDYHUDJHGLVWDQFHEHWZHHQFRQWDFWVZDVVHOHFWHG$OO WKHVWUXFWXUHVZHUHYLVXDOO\LQVSHFWHGZLWKWKH3\0ROYLHZHU VRIWZDUH36
Site-directed mutagenesis of the STE2 gene0XWDWLRQVZHUH
introduced into the receptor gene (STE2) by PCR using the
NLW³4XLN&KDQJH/LJKWQLQJ´ $JLOHQW7HFKQRORJLHV&DW1R
7KHS<(667(SODVPLGZDVXVHGDVWHPSODWHIRU3&5DQG
ZDV FRQVWUXFWHG E\ VXEFORQLQJ WKH ES STE2 gene into WKHS<(6SODVPLG ,QYLWURJHQ&DW1R9 STE2 ZDV
obtained from the pGRB2.2-STE2 plasmid using the XbaI and EcoRI restriction enzymes The pGRB2.2-STE2 plasmid ZDVNLQGO\SURYLGHGE\'UD,UHQH&DVWDxR ,QVWLWXWR3RWRVLQR GH ,QYHVWLJDFLyQ &LHQWt¿FD \ 7HFQROyJLFD 6DQ /XLV 3RWRVt 0p[LFR 0XWDJHQLF SULPHUV ZHUH GHVLJQHG DFFRUGLQJ WR PDQXIDFWXUHU¶V LQVWUXFWLRQV DQG ZHUH FRPSOHPHQWDU\ WR WKH
STE2VHTXHQFHH[FHSWIRUWKH$88FRGRQRI,OH WKDWZDV
FKDQJHGIRU11*&WRLQWURGXFHWKHPXWDWLRQV6HTXHQFHVRI WKHSULPHUVXVHGDUHGHVFULEHGQH[W
1) 5’GCGCTGTTAAAGGTATG11*>GACTTATAAT GATGTTAGTGCCACCC 3’
2) 5’ GGGTGGCACTAACATCATTATAAGTCAC*&11CA TACCTTTAACAGCGC 3’
7KHZKROHYHFWRUS<(667(ZDVDPSOL¿HGXVLQJ3IX7XUEH SRO\PHUDVH7KHQWKH3&5SURGXFWZDVGLJHVWHGZLWK'SQ, UHVWULFWLRQ HQ]\PH DW & IRU PLQXWHV WR HOLPLQDWH WKH SDUHQWDO QRQPXWDWHG SODVPLGDQGWKHQWUDQVIRUPHGLQWR'+Į
E coliVWUDLQ7UDQVIRUPHGFHOOVZHUHVHOHFWHGRQDPSLFLOOLQ
FRQWDLQLQJSODWHVDQGSODVPLGVZHUHLVRODWHGIURPWUDQVIRUPHG FHOOV XVLQJ 4,$SUHS 6SLQ 0LQLSUHS LW 4,$*(1 &DW 1R
,VRODWHGPXWDQWVZHUHFRQ¿UPHGE\'1$VHTXHQFH DQDO\VLV '1$ VHTXHQFLQJ ZDV FDUULHG RXW LQ WKH 8QLGDG GH%LRORJtD0ROHFXODUORFDWHGDWWKH,QVWLWXWRGH)LVLRORJtD
LQIRUPDWLRQ DERXW 6WHS IXQFWLRQ WKDW ZDV QRW QHFHVVDULO\
incorporated in previous models of this interaction; our model
ZDV EXLOW XVLQJ WKH VWDWH RI DUW WRROV XVHG WR EXLOG WKUHH
GLPHQVLRQDOPRGHOVRISURWHLQV,QRXUPRGHO,OH LQ6WHSD
FRQVHUYHGSRVLWLRQDPRQJIXQJDOVSHFLHVGRHVQRWLQWHUDFWZLWK
WKHSKHURPRQH7RWHVWRXUPRGHOZHSHUIRUPHGDPXWDJHQHVLV
at this residue We choose this residue because it is part of the
VHFRQGH[WUDFHOOXODUORRSZKLFKKDVEHHQSUHYLRXVO\LPSOLHGWR
play a role in ligand recognition and binding in other GPCRs,
such as the dopamine D221 07 PHODWRQLQ22, thyrotropin23
angiotensin24, and histamine H125 receptors Our results indicate
WKDWVRPHPXWDQWVRIWKLVUHVLGXHKDYHDVPDOOH൵HFWRQWKH
6WHSVLJQDOLQJFDVFDGHWKDWOHDGVFHOOVWRDUUHVWFHOOJURZWK
in the presence of the pheromone The structural bases of these
results are discussed using our three-dimensional model
MATERIAL AND METHODS
The construction of a Ste2 model was done using the rhodopsin
crystal as a template.7KHUHFHSWRUPRGHOZDVEXLOWXVLQJWKH
L7$66(59VHUYHU26L7$66(5ZDVFKRVHQEHFDXVHLWZDV
UDQNHGDVWKHVHUYHUZLWKEHVWUHVXOWVLQ&$63WR&$63
(Critical Assessment of Techniques for Protein Structure
3UHGLFWLRQ :HPRGHOHGUHVLGXHVWR 7KH1WHUPLQDO
UHVLGXHV DQG &WHUPLQDO GRPDLQV UHVLGXHV
ZHUHQRWPRGHOHGEHFDXVHWKHVHGRPDLQVDUHQRWHVVHQWLDOIRU
ligand binding
'HVSLWHWKHIDFWWKDW6WHSDQGUKRGRSVLQKDYHDORZVHTXHQFH
LGHQWLW\ UKRGRSVLQ ZDV XVHG DV D WHPSODWH IRU 6WHS
PRGHOLQJEHFDXVH 6WHSDQGUKRGRSVLQVKDUHVWUXFWXUDODQG
functional properties, 2) available structural and mutational data
VKRZWKDWWKH6WHSDQGUKRGRSVLQVHTXHQFHVDOLJQSURSHUO\
based on a comparison of amino acids that have similar structural
and functional roles in membrane proteins and 3) rhodopsin
has been used as a template for previously developed Ste2p
models7KHVHTXHQFHVIRU6WHSDQGUKRGRSVLQZHUHREWDLQHG
from the UniProt database The crystal structure of rhodopsin
3'%HQWU\FRGH8 ZDVREWDLQHGIURPWKH3URWHLQ'DWD
Bank 7KHDOLJQPHQWRI6WHSZLWKUKRGRSVLQZDVFDUULHGRXW
as described previously
7KHPHPEUDQHERXQGDULHVLQWKH6WHSVHTXHQFHZHUHREWDLQHG
from solvent accessibility data20DQGZHUHXVHGLQL7$66(5WR
specify boundaries of secondary structures To guide i-TASSER
RQ WKH FRUUHFW KHOL[ RULHQWDWLRQ GLVWDQFH UHVWUDLQWV EHWZHHQ
VSHFL¿FUHVLGXHVZHUHREWDLQHGIURPWKH3'%¿OHRIUKRGRSVLQ
XVLQJ WKH GLVWDQFH EHWZHHQ WKH DOSKDFDUERQ IURP D UHVLGXH
placed at the end of a helix and the alpha-carbon from the
residues localized at the ends of all the other helices
Construction of the alpha-pheromone three-dimensional
model 7KH3(3)2/'VHUYHU30ZDVXVHGWRGHYHORSDQDWRPLF
three-dimensional model of the alpha pheromone peptide
7KH VHTXHQFH RI DOSKD SKHURPRQH 7US+LV7US/HX*OQ
Trang 4&HOXODU&RQVWUXFWVZHUHWUDQVIRUPHGLQWRDSTE2 null mutant
0$7DVWUDLQ %<¨<)/2: NDQ0; DVSUHYLRXVO\
GHVFULEHGHOVHZKHUHDQGWUDQVIRUPHGFHOOVZHUHVHOHFWHGE\
WKHLUJURZWKLQPLQLPDOPHGLXPODFNLQJXUDFLO
Growth curves Yeast cultures expressing the mutant receptor
ZHUHLQFXEDWHGIRUKRXUVDW&ZLWKPLQLPDOPHGLXP
ODFNLQJ XUDFLO ZLWK JOXFRVH DQG WKHQ ZHUH LQFXEDWHG IRU
DGGLWLRQDO KRXUV ZLWK PLQLPDO PHGLXP ODFNLQJ XUDFLO ZLWK
galactose in order to induce the expression of STE2 in the plasmid
$IWHUZDUGV\HDVWFHOOVZHUHGLOXWHGWRRSWLFDOGHQVLW\RI
DQGLQFXEDWHGZLWKSKHURPRQH $QDVSHF DW0LQWRD
ZHOOKDOIDUHDSODWHLQDWRWDOYROXPHRIO7KHLQFXEDWLRQ
ZDVFDUULHGRXWDW&ZLWKVKDNLQJDQGWKH$QPZDV
PHDVXUHGHYHU\KRXUIRUKXVLQJD6\QHUJ\0;SODWHUHDGHU
%LR7HN,QVWUXPHQWV 7KHH[SHULPHQWZDVUHSHDWHGWKUHHWLPHV
HDFKWLPHZLWKDWULSOLFDWH*URZWKFXUYHVGDWDZHUHQRUPDOL]HG
DQGWKHDUHDXQGHUWKHFXUYHZDVFDOFXODWHGIRUHDFKPXWDQW
RESULTS AND DISCUSSION
Three-dimensional model of Ste2p.7KH6WHSPRGHOZLWKWKH
EHVW&VFRUH FRQ¿GHQFHVFRUH ZDVVHOHFWHG )LJXUH LQRXU
FDVHWKH&VFRUHZDVDQGWKH70VFRUHZDV7KH C-score given by i-TASSER estimates the quality of predicted PRGHOVLVLQWKHUDQJHRI>@0RGHOVZLWK&VFRUH!WHQG WREHFORVHWRWKHQDWLYHVWUXFWXUH7KH70VFRUHSURYLGHGE\ i-TASSER estimates the similarity of the protein model and its WHPSODWH DVFRUHEHORZFRUUHVSRQGVWRWZRSURWHLQVWKDW DUHQRWVLPLODUZKLOHDERYHFRUUHVSRQGWRSURWHLQVWKDWDUH VWUXFWXUDOO\KRPRORJXHV7KXVWKH70VFRUHLQGLFDWHVWKH6WHS PRGHOGL൵HUVIURPWKHUKRGRSVLQWHPSODWH 8 DVH[SHFWHG VHH0HWKRGV EXWUHPDLQVVLPLODU,QRXUWKUHHGLPHQVLRQDO model the boundaries of the transmembrane helices matched the solvent accessibility data previously described , except for WKH+7KLVKHOL[KDVEHHQUHSRUWHGWRH[SDQGIURP/HXWR /HX )LJXUH ZKLOHLQRXUPRGHOLWVSDQVIURP3KHWR ,OH7KLVGL൵HUHQFHUHÀHFWVWKHIDFWWKDWWKHORRSFRQQHFWLQJ +WR+SUHVHQWVWZRUHVLGXHV DQG DWWKHHQGRIWKLV ORRSWKDWDUHQRWDFFHVVLEOHWRELRWLQ\ODWLRQIROORZHGE\WZR RWKHUUHVLGXHVWKDWDUHDFFHVVLEOHWRELRWLQ\ODWLRQ DQG 7KHDXWKRUVSURSRVHGWKDW+VKRXOGVWDUWDWUHVLGXHEXW ZHSUHIHUWRXVHWKH¿UVWUHVLGXHVWKDWZHUHLQDFFHVVLEOHDVSDUW
of this helix These authors could not establish the end of this KHOL[VRZHXVHGDVDFULWHULRQWKHVHTXHQFHDOLJQPHQWEHWZHHQ 6WHSDQGUKRGRSVLQJHQHUDWHGZLWKL7$66(5
7KH 6WHS PRGHO VKRZHG KHOL[KHOL[ LQWHUDFWLRQV WKDW ZHUH FRQVLVWHQW ZLWK ELRFKHPLFDO GDWD )RU H[DPSOH 6WHS KDV D GXXXG motif in H1 that includes Gly56 and Gly60 These motifs mediate helix-helix interactions and have been implicated in the dimerization of Ste2p10 To achieve this, these JO\FLQHVPXVWIDFHRXWZDUGVWKHSURWHLQ,QRXUPRGHOWKHVH glycines are properly oriented (see Figure 3)
$V RSSRVHG WR JOREXODU SURWHLQV LQWHUDFWLRQV EHWZHHQ transmembrane helices are generally mediated by polar amino acids located in the transmembrane region These amino acids can form single helix-helix contacts or hydrogen-bonding QHWZRUNV,WKDVEHHQSURSRVHGEDVHGRQVHTXHQFHFRQVHUYDWLRQ that the H1-H2 interaction is mediated by a hydrogen bond EHWZHHQ$UJ +, DQG+LV + +RZHYHULQRXU6WHS PRGHO$UJPD\IRUPDK\GURJHQERQGZLWK7\URQWKH same face of H2 (see Figure 3), so further mutagenesis and structural experiments may test for the relevance of these pairs
of residues in the H1-H2 interaction Similarly, our model SURSRVHGRWKHUQHZKHOL[KHOL[LQWHUDFWLRQV)RULQVWDQFHWKH SUR[LPLW\RIUHVLGXHV6HU + DQG*OX + ZRXOG PHGLDWH++LQWHUDFWLRQV VHH)LJXUH /LNHZLVH6HU DQG7KURIWKHH[WUDFHOOXODUORRSPD\LQWHUDFWZLWK*OQ (H1) (see Figure 3) Our three-dimensional model of Ste2p also GLVSOD\VWKHRULHQWDWLRQRIUHVLGXHVWKDWDUHFRQVLVWHQWZLWKWKHLU IXQFWLRQDOUROHLQRWKHU*3&5V)RULQVWDQFHZHREVHUYHGWKDW
*OQ DSSHDUVWREHIXQFWLRQDOO\HTXLYDOHQWWR*OXZKLFK
is part of the ERY motif in rhodopsin Both Glu134 in rhodopsin DQG*OQ LQ6WHSDUHORFDOL]HGLQDVLPLODUSRVLWLRQDWWKH HQG RI + DQG DUH UHVLGXHV ZKHUH PXWDWLRQ WR DUJLQLQH RU
Figure 2 Three-dimensional structural model of Ste2p A
ribbon representation of the three-dimensional structure of
Ste2p is presented Ste2p has a central core made of seven
transmembrane helices (HI to H7) connected by three
intracellular (IL1, IL2 and IL3) and three extracellular loops
(EL1, EL2 and EL3) This model shows the counterclockwise
orientation of transmembrane helices used for modeling
GPCRs The image was generated with PyMol.
Trang 5alanine leads to constitutive activation40-42,QDJUHHPHQWZLWK
WKHVHSUHYLRXVREVHUYDWLRQV*OQ LVRULHQWHGLQDSRVLWLRQ
similar to that of Glu134 in rhodopsin and presumably shares
the role of preventing constitutive activation of the receptor;
the reported single point mutations to alanine in positions
*OQ RU $VQ UHQGHUHG D FRQVWLWXWLYH DFWLYDWLRQ RI WKH
6WHSUHFHSWRUDQGDGRXEOHPXWDWLRQWKDWVZDSSHGWKHVLGH
FKDLQVRIWKHVHWZRUHVLGXHVPDLQWDLQHGDZLOGW\SHSKHQRW\SH
suggesting these residues are in close proximity in the 3D
VWUXFWXUHRI6WHS,QRXUPRGHO*OQ DQG$VQDUH
cDSDUWDQGIDFLQJDZD\IURPHDFKRWKHULQGLFDWLQJWKDWRXU
PRGHOLVQRWFRQVLVWHQWZLWKWKHVHH[SHULPHQWDOUHVXOWV6LQFH
RXUPRGHOGLGQRWLQFOXGHWKH1DQG&WHUPLQLORFDWHGDWWKH
VLGHZKHUH*OQ OLHVLWVHHPVWKDWWKHRULHQWDWLRQRI+DQG
H3 on this side of the transmembrane region of the receptor are
not properly oriented in our model Yet, pheromone binding
takes place at the opposite side of the transmembrane region
DQGSUHVXPDEO\GRHVQRWLQFOXGHWKHVHKHOLFHVVRZHSURFHHG
to analyze the reliability of our model
Docking7KHOLJDQGELQGLQJRULHQWDWLRQVYDU\ZLGHO\EHWZHHQ
the members of the GPCR family43 ,W KDV EHHQ VKRZQ WKDW
ODUJHOLJDQGVELQGWRWKHH[WUDFHOOXODUORRSVRI*3&5VZKLOH
small ligands bind to the transmembrane region of receptor
In the case of peptide ligands it has been proposed that a
combination of both binding modes takes place In such case, WKHOLJDQGELQGV¿UVWWRH[WUDFHOOXODUORRSVDQGODWHUHQWHUVWKH WUDQVPHPEUDQHGRPDLQZKHUHWKHELQGLQJSRFNHWDFWVOLNHDQ epicenter of conformational changes These conformational changes are propagated to intracellular loops through the movement of helices Then, the intracellular domain binds to and activates the trimeric G protein43 In the case of Ste2p, alpha pheromone binding has been mapped to the pocket formed by WKHH[WUDFHOOXODUHQGVRIWKHWUDQVPHPEUDQHKHOLFHVVSHFL¿FDOO\ H1, H5, and H6
7KH *3&5V LQWHUDFW ZLWK WKHLU OLJDQGV YLD K\GURJHQ ERQGV ionic pairs and hydrophobic contacts447KHLQWHUDFWLRQVEHWZHHQ Ste2p and the alpha pheromone responsible for binding and DFWLYDWLRQ DUH QRW ZHOO FKDUDFWHUL]HG +RZHYHU HOHFWURVWDWLF interactions have been suggested to play a critical role in the Ste2p-alpha pheromone complex45
The docking simulation aimed to model the Ste2p-alpha pheromone interaction (Figure 4) In this model all contacts
in the Ste2p-alpha pheromone interaction previously reported DUHSUHVHQW)RULQVWDQFHSUHYLRXVVWXGLHVDERXWWKHD൶QLWLHV and activities of various alpha pheromone analogues have VKRZQ WKDW UHVLGXHV 6HU DQG7KU DUH QHDU *OQ LQ WKH alpha pheromone$VZHVHHLQ)LJXUHRXUELQGLQJPRGHOLV
Figure 3 Residues involved in helix-helix interactions in Ste2p model The transmembrane helices of Ste2p are presented as ribbons and are enumerated from H1 to H7 For convenience, transmembrane helices are displayed in a clockwise orientation Hydrogen bonds are formed between Arg58-Tyr101, Ser170-Glu143, and Ser107-Gln51-Thr110, and are displayed with dots Gly56 and Gly60 are oriented outwards the protein The image was generated with PyMol.
Trang 6extracellular ends of the helices H5 and H6, respectively These
helices are linked at the opposite end involved in pheromone
ELQGLQJE\WKHWKLUGLQWUDFHOOXODUORRSWKDWLQWHUDFWVZLWKWKH
trimeric G protein and promotes its activation Site-directed
PXWDJHQHVLV VWXGLHV VKRZHG WKDW 3KH LQWHUDFWV ZLWK WKH
&WHUPLQDOUHJLRQRIWKHSKHURPRQH OLNHO\ZLWK7\U ZKLOH
7\ULQWHUDFWVZLWKWKH1WHUPLQDOUHJLRQ OLNHO\ZLWK7US
or Trp3) Both residues, Phe204 and Tyr266, play roles in the transformation of Ste2p into an activated state upon agonist binding$VVKRZQLQ)LJXUHRXUPRGHOLVFRQVLVWHQWZLWK WKHVHH[SHULPHQWDOGDWDGHVSLWHWKHSUHYLRXVO\QRWHGGL൵HUHQFHV
in our model about H6 and the intracellular ends of H2 and + UHVLGXHV$VQDQG*OQ )LQDOO\LQWHUDFWLRQEHWZHHQ
Figure 4 Atomic three-dimensional model of the interaction between Ste2p and the alpha pheromone Ste2p is shown in a light JUH\ULEERQUHSUHVHQWDWLRQ7KHDOSKDSKHURPRQHLVVKRZQDVDGDUNJUH\REMHFW7KHXSSHUSDUWRIWKHÀJXUHLOOXVWUDWHVWKH ELQGLQJSRFNHWIRUPHGE\H[WUDFHOOXODUORRSVDQGH[WUDFHOOXODUHQGVRI++DQG+,QWKHORZHUSDUWRIWKHÀJXUHVHOHFWHG residues of Ste2p and pheromone are shown in a ball-and-stick representation First, the C-terminal region of the pheromone (Gln10) binds to the receptor residues Ser47 and Thr48 (H1) 17 Then, the central region of the pheromone (Pro8-Gly9) forms a ǃWXUQ 46 At this point, Phe204 (H5) is near to Tyr13 in the pheromone 18 , and Lys269 (H6) is near to both the N and C-termini of the pheromone Asn205 is facing toward the outside of Ste2p and away from the binding pocket (left) In our model, Tyr266 (H6) is oriented towards the surrounding lipids and is inaccessible to solvent This orientation is consistent with experimental data from 18 Finally, Tyr266 interacts with Trp3 in the pheromone, mediating the transition to an active state of Ste2 18,47 (right) The image was generated with PyMol.
Trang 7aromatic residues in the alpha pheromone and Ste2p could be
VWDELOL]HGE\ULQJVWDFNLQJH൵HFWVRUE\K\GURJHQERQGLQJ,Q
RXUPRGHOWKHLQWHUDFWLRQEHWZHHQ7\UDQG7USLVVWDELOL]HG
by hydrogen bonding (Figure 4)
+RZHYHURXUPRGHOGRHVQRWSUHVHQWWKH$VQSKHURPRQH
contact Asn205 has been implicated in the binding of the
1WHUPLQDO GRPDLQ RI WKH SKHURPRQH EHFDXVH PXWDWLRQV DW
WKLV SRVLWLRQ D൵HFW WKH 6WHS ELQGLQJ WR WKH SKHURPRQH33 It
KDVEHHQVKRZQWKDWUHVLGXHVWKDWDUHLPSRUWDQWIRUELQGLQJDUH
not necessarily near the ligand or at the interface in the
three-dimensional structure ,QRXU6WHSPRGHO$VQIDFHVDZD\
from the binding pocket (Figure 4) Thus, according to our model,
$VQZRXOGFRQWULEXWHWRELQGLQJQRWE\GLUHFWLQWHUDFWLRQ
ZLWKWKHSKHURPRQH,QVXFKFDVHLWZRXOGEHH[SHFWHGWKDW
Asn205 may contribute to the pheromone binding through a
VHWRILQWHUDFWLRQVZLWKUHVLGXHVWKDWVXSSRUWWKHSRVLWLRQRI
the residues that directly bind to the pheromone Analyzing the
FRQWDFWV FXWR൵GLVWDQFH c RIWKH6WHSUHVLGXHVLQYROYHG
LQWKHSKHURPRQHELQGLQJ 6HU7KU3KHDQG7\U
DQG FRPSDULQJ WKHVH ZLWK WKH FRQWDFWV RI UHVLGXH $VQ
ZHOHDUQHGWKHVHVKDUH UHVLGXHV 9DO 7\U $VQ
7\U3KH$OD6HU7KUDQG,OH ZKLFK
FRUUHVSRQGVWR RIWKHUHVLGXHVWKDWVXSSRUWWKHSRVLWLRQ
of the residues directly binding the pheromone This analysis
further supports the relevance of Asn205 in pheromone binding
WKURXJK LQGLUHFW FRQWDFWV ZLWK WKH OLJDQG ELQGLQJ UHVLGXHV
These results constitute a hypothesis that can be experimentally
WHVWHGE\SHUIRUPLQJGRXEOHVZDSPXWDQWVDW$VQDQGWKH
UHVLGXHVLQWKHSKHURPRQHSURSRVHGWRLQWHUDFWZLWK$VQ
IRULQVWDQFH LQWKHSUHVHQWVWXG\ZHGHFLGHGWRPXWDWHDQRWKHU
UHJLRQRIWKHSURWHLQDVH[SODLQHGEHORZ
Additionally, our three-dimensional model has some
GLVFUHSDQFLHV ZLWK RWKHU FRPSXWDWLRQDO VWXGLHV UHSRUWHG
SUHYLRXVO\)RUH[DPSOH3KHGRHVQRWLQWHUDFWZLWK7\U
of the alpha pheromone in the Umanah model34 The lack of
LQWHUDFWLRQ EHWZHHQ WKHVH UHVLGXHV ZDV PRGHOHG EDVHG RQ
studies of alpha pheromone analogues and chemical
cross-OLQNLQJ H[SHULPHQWV WKDW SURSRVHG DQ LQWHUDFWLRQ EHWZHHQ
7\U RI WKH DOSKD SKHURPRQH ZLWK $UJ RI 6WHS Our
PRGHO RQ WKH RWKHU KDQG LQFOXGHV WKH LQWHUDFWLRQ EHWZHHQ
7\UDQG3KHSURSRVHGE\/HHDQGFROOHDJXHV, based on
site-directed mutagenesis and cross-linking experiments that
ZHUHGLVUHJDUGHGE\WKHJURXSRI8PDQDKDQGFROOHDJXHV:H
prefer to use the Tyr13-Phe204 interaction in the construction of
our Ste2p model because it has more support by other research
groupsWKDQWKH7\U$UJLQWHUDFWLRQ
,Q WKH LQWHUIDFH ZH IRXQG UHVLGXHV ERWK DFFHVVLEOH DQG
inaccessible to solvent This is a feature of many other interfaces
RI SURWHLQV ZKHUH VROYHQW DFFHVVLEOH UHVLGXHV SOD\ UROHV LQ
OLJDQGELQGLQJZKLOHWKRVHQRWDFFHVVLEOHWRVROYHQWPHGLDWH
the transition to an active state52 Additionally, our binding
PRGHODOORZVXVWRSURSRVHWKDWWKHLQWHUIDFHEHWZHHQ6WHS DQGWKHSKHURPRQHLVFRPSRVHGRIUHVLGXHV$VVKRZQLQ Table I, interface residues include both polar and non-polar UHVLGXHV7KLVLVFRQVLVWHQWZLWKSUHYLRXVVWXGLHVLQGLFDWLQJWKDW both hydrophobic and hydrophilic residues form the binding pocket of Ste2p53 The interface residues that are structurally and functionally important tend to be conserved residues or WKH\KDYHDORZHUUDWHRIPXWDWLRQFRPSDUHGZLWKWKHUHVWRI the residues of the protein547KLVLVWKHFDVHIRU7\UZKLFK LVDFULWLFDOUHVLGXHIRUELQGLQJDVZHOODVVLJQDOWUDQVGXFWLRQ DQGVKRZVDODUJHSHUFHQWDJHRIVHTXHQFHLGHQWLW\FRQVHUYDWLRQ VHH7DEOH, +RZHYHUWKLVLVQRWDOZD\VWKHFDVHDQGPDQ\ residues that are important for binding are not conserved55 ,QDJUHHPHQWZLWKWKLVLGHD7DEOH,VKRZVWKDWIHZLQWHUIDFH UHVLGXHV DUH FRQVHUYHG DPRQJ 6WHS RUWKRORJXHV 9DO 6HU/HX7KU7\U )XUWKHUPRUH6HUDQG 7KURIWKH¿UVWH[WUDFHOOXODUORRSIRXQGLQWKHLQWHUIDFHRI 6WHDQGWKHDOSKDSKHURPRQHLQWKLVVWXG\ZHUHSUHYLRXVO\ proposed as critical for signaling16*OQDQG6HUDOVR found at the interface, are hydrogen bonded as suggested by WKH6WHSPRGHOGHYHORSHGLQWKLVZRUN\HWWKHVHSRVLWLRQV
do not preserve the same side chain as Ste2p in other fungal RUWKRORJXHV&RQVLGHULQJWKDWWKH¿UVWH[WUDFHOOXODUORRSKDV been observed to undergo a conformational change upon ligand binding15 WKLV K\GURJHQ ERQG FRXOG EH PRGL¿HG GXULQJ WKH
signaling process Other non-conserved residues (e.g9DO
$VS ZHUH DOVR IRXQG LQ WKH LQWHUIDFH WKDW FRXOG SOD\ D critical role in Ste2p function Site-directed mutagenesis on these residues may help to test the role of these residues in ligand binding or signaling
Site-directed mutagenesis of STE26HYHUDOVWXGLHVKDYHVKRZQ
that the second extracellular loop of GPCRs plays an important role in ligand binding21-25 Since no mutation for Ste2p at this ORRSKDVEHHQUHSRUWHGZHGLGQRWXVHWKLVLQIRUPDWLRQWRJXLGH the docking of the pheromone to Ste2p A sequence conservation DQDO\VLV SHUIRUPHG DPRQJ IXQJDO UHFHSWRUV KWWS ZZZ
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hese results indicate that this position is not critical for receptor function, but ring and positively-charged side chains are tolerated OHVVDWWKLVSRVLWLRQ,QRXU6WHSDOSKDSKHURPRQHPRGHO,OH LVRULHQWHGDZD\IURPWKHELQGLQJSRFNHWEHKLQG/\VD direct ligand of the pheromone (Table I) Placing an arginine DWSRVLWLRQ FRXOGUHVXOWLQDQH[FHVVRISRVLWLYHFKDUJHDW
Trang 8WKLVVLWHDOWHULQJWKHFRQIRUPDWLRQRI/\VDQGGHELOLWDWLQJ WKHLQWHUDFWLRQZLWKWKHSKHURPRQH3ODFLQJDQRWKHUULQJVXFK DV 3UR RU +LV DW SRVLWLRQ FRXOG PRGLI\ WKH SRVLWLRQ RI 7\UDQRWKHUUHVLGXHLQGLUHFWFRQWDFWZLWKWKHSKHURPRQH DQGRULQWHUDFWZLWK+LVDWWKH1WHUPLQXVRI+ )LJXUH
DOWHULQJWKHWLOWRI+ZLWKDOORVWHULFFRQVHTXHQFHVDWWKH cytoplasmic loops that could impair signaling
9DOLGDWLQJ D PRGHO UHTXLUHV WHVWLQJ IRXU GL൵HUHQW VWDWLVWLFDO SDUDPHWHUV WUXH SRVLWLYH 73 WUXH QHJDWLYH 71 IDOVH SRVLWLYH )3 DQGIDOVHQHJDWLYH )1 SUHGLFWLRQV<HWPRVW modeling studies of protein structure commonly test for TP; IRULQVWDQFHWKHZRUOGFRQWHVWRQSURWHLQVWUXFWXUHSUHGLFWLRQ only tests for TP by establishing a score that accounts for the fraction of the model that may be superimposed to the real SURWHLQVWUXFWXUHZLWKQRPRUHWKDQFHUWDLQFXWR൵506'YDOXH56 The relevance of the other 3 parameters becomes apparent ZKHQLWLVUHFRJQL]HGWKDWH[SHULPHQWDOSURWHLQ'VWUXFWXUHV should also be considered a model to test for the critical role RIUHVLGXHVLQSURWHLQIXQFWLRQ,QWKHSUHVHQWZRUNZHWHVWHG
4 parameters in our model, thus providing a more complete test about the quality of the generated structure Particularly, ZHVKRZHGWKDW
Figure 5 Area under the growth curve for each mutant strain
7KHSRSXODWLRQJURZWKRI\HDVWVWUDLQVH[SUHVVLQJDQ\RIÀYH
mutants at Ile190 (I190L, I190S, I190R, I190H and I190P) is
represented by the Area Under the growth curve (Y axis) both in
the presence and absence of the alpha pheromone (indicated
E\WKHDEEUHYLDWLRQ´SKHUµLQWKHÀJXUH $VFRQWUROWKHJURZWK
of wild type strain (BY4741) and a strain carrying a deletion of
STE2 gene are presented.
H1 Extracellular loop 1 Extracellular loop 2 and H5 Extracellular loop 3 and H6 Ser47
Accessible to solvent
Ser107
(16%) Accessible to solvent
Asn194
Ala265
Inaccessible to solvent
Thr48
Accessible to solvent
Ser108 (27%)
Asp195
(12%)
Tyr266 (45%)
Inaccessible to solvent
Val49
(32%)
Accessible to solvent
Leu113 (26%)
Val196
(13%)
Ser267
(10%) Inaccessible to solvent
Thr50
Inaccessible to solvent
Thr114 (30%)
Gln200
Accessible to solvent
Lys269
(3%) Accessible to solvent
Gln51
unaccessible to solvent
Phe116
(12%)
Asp201
(5%) Accessible to solvent
Pro270
(5%) Accessible to solvent
Met54
(6%)
Inaccessible to solvent
Pro117
(11%)
Lys202
Accessible to solvent
Gly273
Accessible to solvent
Tyr203
Accessible to solvent
Phe204
(10%) Accessible to solvent
7DEOH,&RPSRVLWLRQRIWKHLQWHUIDFHEHWZHHQ6WHDQGDOSKDSKHURPRQH,QWHUIDFHUHVLGXHVZHUHGH¿QHGDVWKRVHUHVLGXHVLQ6WHSWKDWZHUH
no more than 5 Å apart from the pheromone Solvent accessibility data were obtained from Lin, J.C., et al., 200453 Percentage of conservation (indicated in parenthesis for each residue; this percentage represents the fraction of 214 orthologue sequences that have an identical residue to Ste2p at that position) was derived from the multiple sequence alignment for all the Ste2p orthologues reported for PFAM family PF02116 Those positions with the largest percentage of sequence identity conservation are marked in bold.
Trang 9TP are residues predicted critical and indeed are critical for
SURWHLQIXQFWLRQVWUXFWXUH)RULQVWDQFHZHVKRZWKDWUHVLGXHV
Phe204 and Tyr266 that are close to the pheromone in our model
are indeed important for binding
FP are residues predicted critical but are not truly relevant for
SURWHLQIXQFWLRQVWUXFWXUH+HUHZHVKRZWKDWUHVLGXH,OH LV
predicted by sequence conservation to be relevant for protein
VWUXFWXUHIXQFWLRQ\HWRXUH[SHULPHQWDOUHVXOWVSURYLGHHYLGHQFH
against this hypothesis
71DUHUHVLGXHVSUHGLFWHGQRWFULWLFDODQGDUHQRWWUXO\UHOHYDQWIRU
SURWHLQIXQFWLRQVWUXFWXUH2XU'PRGHOSUHGLFWHGWKDW,OH
ZDVQRWFULWLFDODQGKHUHZHVKRZWKDWLQGHHGWKLVLVWKHFDVH
)1 DUH UHVLGXHV WKDW DUH SUHGLFWHG QRW FULWLFDO EXW DUH WUXO\
UHOHYDQWIRUSURWHLQIXQFWLRQVWUXFWXUH2XUPRGHOSUHGLFWVWKDW
UHVLGXHV*OQ DQG$VQDUHIDUDSDUWIURPHDFKRWKHU\HW
H[SHULPHQWDOHYLGHQFHKDVVKRZQRWKHUZLVH
1RWHWKDW71DQG)1DUHGL൶FXOWWRWHVWLQSURWHLQVWUXFWXUH
IXQFWLRQ VWXGLHV EHFDXVH LQ PRVW FDVHV RQO\ UHVLGXHV ZLWK
DQH൵HFWRQSURWHLQVWUXFWXUHRUIXQFWLRQDUHUHSRUWHGLQWKH
OLWHUDWXUH<HWZHDUJXHWKLVLQIRUPDWLRQLVSDUWLFXODUO\UHOHYDQW
in testing for structure-function prediction models, such as in
WKHFDVHRIUHVLGXHVOLNH,OH
CONCLUSIONS
The present study presents an atomic three-dimensional structure
model of the alpha pheromone receptor from S cerevisiae,
Ste2p, based on state-of-the-art modeling methods and current
DYDLODEOHH[SHULPHQWDOGDWD7KH6WHSPRGHOLVFRQVLVWHQWZLWK
PRVWDYDLODEOHELRFKHPLFDOLQIRUPDWLRQDQGDOORZVXVWRSURSRVH
VSHFL¿FLQWHUDFWLRQVWKDWFRXOGVWDELOL]HWKHQDWLYHFRQIRUPDWLRQ
RIWKHUHFHSWRU)XUWKHUPRUHDPRGHORIWKHLQWHUDFWLRQEHWZHHQ
6WHSDQGWKHDOSKDSKHURPRQHZDVJHQHUDWHGEDVHGRQZKDWLV
NQRZQDERXWWKHELQGLQJFRQWDFWV7KH6WHSDOSKDSKHURPRQH
PRGHODOORZHGXVWRLGHQWLI\UHVLGXHVWKDWDUHSUHVXPDEO\
IRXQGLQWKHLQWHUIDFHEXWGLGQRWLQFOXGH,OH DFRQVHUYHG
UHVLGXHVDPRQJIXQJLVSHFLHV0XWDJHQHVLVRI,OH LQ6WHS
KDGVPDOORUQRH൵HFWRQUHFHSWRUIXQFWLRQDQGLQWKHPRGHOWKLV
UHVLGXHSRLQWVDZD\IURPWKHSKHURPRQH7KXVWKHPRGHOPD\
serve as a starting point for further site-directed mutagenesis to
test the structure-function relationship of this receptor
7KLV ZRUN ZDV LQ SDUW VXSSRUWHG E\ 3$3,,7 JUDQW QXPEHU
,1WR*DEULHOGHO5tR/DXUD0DULQD5REOHV IHOORZVKLS
QXPEHU ZDVVXSSRUWHGLQSDUWE\&RQVHMR1DFLRQDOGH
&LHQFLD\7HFQRORJtD &21$&<7 ZKLOHVKHZDVVWXGHQWRI
WKH3URJUDPDGH0DHVWUtDHQ&LHQFLDV%LRTXtPLFDV81$0
:HWKDQN0DUtD7HUHVD/DUD2UWL]DWWKH,QVWLWXWHRI&HOOXODU
3K\VLRORJ\8QLYHUVLGDG 1DFLRQDO$XWyQRPD GH 0p[LFR IRU
KHUWHFKQLFDODVVLVWDQFHLQWKLVZRUN
REFERENCES
1 /XQGVWURP $Q RYHUUHYLHZ RQ *3&5V DQG GUXJ GLVFRYHU\ structure-based drug design and structural biology on GPCRs
Methods Mol Biol. '2,
B
2 5RVHQEDXP'05DVPXVVHQ6*) .RELOND%.7KHVWUXFWXUH
and function of G-protein-coupled receptors Nature, 459(7245),
'2, QDWXUH
3 0DFND\9 0DQQH\750XWDWLRQVD൵HFWLQJVH[XDOFRQMXJDWLRQ
and related processes in Saccharomyces cerevisiae I Isolation and phenotypic characterization of nonmating mutants Genetics,
76(2)
4 +DUWZHOO/+0XWDQWVRISaccharomyces cerevisiae unresponsive
to cell division control by polypeptide mating hormone J Cell
Biol 85±
5 -HQQHVV '' %XUNKROGHU$& +DUWZHOO /+ %LQGLQJ RI ĮIDFWRUSKHURPRQHWR\HDVWDFHOOV FKHPLFDODQGJHQHWLFHYLGHQFH
IRUDQĮIDFWRUUHFHSWRUCell, 35(2)
6 %XUNKROGHU$& +DUWZHOO/+7KH\HDVWĮIDFWRUUHFHSWRU Structural properties deduced from the sequence of the STE2 gene
Nucleic Acids Res 13±
1HXPRLQ$ &RKHQ /6$UVKDYD % 7DQWU\ 6 %HFNHU - 0=HUEH2 1DLGHU)6WUXFWXUHRIDGRXEOHWUDQVPHPEUDQH
fragment of a G-protein-coupled receptor in micelles Biophysical
Journal, 96(8) '2, MESM
9DOHQWLQH.*/LX6)0DUDVVL)09HJOLD*2SHOOD6-'LQJ ); 1DLGHU)6WUXFWXUHDQGWRSRORJ\RIDSHSWLGHVHJPHQWRIWKH
6th transmembrane domain of the Saccharomyces cerevisae
alpha-factor receptor in phospholipid bilayers Biopolymers, 59(4),
243-256 (2001) '2, $,'
%,3!&2 +
8GGLQ 06 LP + 'H\R $ 1DLGHU ) %HFNHU -0 ,GHQWL¿FDWLRQRIUHVLGXHVLQYROYHGLQKRPRGLPHUIRUPDWLRQORFDWHG ZLWKLQDȕVWUDQGUHJLRQRIWKH1WHUPLQXVRID<HDVW*SURWHLQ
coupled receptor Journal of Receptors and Signal Transduction
Research 32(2) '2,
10 2YHUWRQ0& %OXPHU.-7KHH[WUDFHOOXODU1WHUPLQDOGRPDLQ and transmembrane domains 1 and 2 mediate oligomerization of a
yeast G protein-coupled receptor Journal of Biological Chemistry,
277(44) '2, MEF0
11 6FKDQGHO.$ -HQQHVV'''LUHFWHYLGHQFHIRUOLJDQGLQGXFHG
LQWHUQDOL]DWLRQRIWKH\HDVWĮIDFWRUSKHURPRQHUHFHSWRUMol Cell
Biol 14
12 +LFNH / 5LH]PDQ + 8ELTXLWLQDWLRQ RI D \HDVW SODVPD
membrane receptor signals its ligand-stimulated endocytosis Cell
84(2)
13 5RKUHU-%HQHGHWWL+=DQRODUL% 5LH]PDQ+,GHQWL¿FDWLRQ
of a novel sequence mediating regulated endocytosis of the G
SURWHLQFRXSOHGĮSKHURPRQHUHFHSWRULQ\HDVWMol Biol Cell
4
14 6WHIDQ&- %OXPHU.-7KHWKLUGF\WRSODVPLFORRSRID\HDVW
*SURWHLQFRXSOHG UHFHSWRU FRQWUROV SDWKZD\ DFWLYDWLRQ OLJDQG
discrimination, and receptor internalization Mol Cell Biol 14(5),
15 +DXVHU0.DX൵PDQ6/HH%.1DLGHU) %HFNHU-0 7KH ¿UVW H[WUDFHOOXODU ORRS RI WKH 6DFFKDURP\FHV FHUHYLVLDH * protein-coupled receptor Ste2p undergoes a conformational change
upon ligand binding Journal of Biological Chemistry, 282(14),
Trang 1016 $NDO6WUDGHU$ KDUH 6 ;X ' 1DLGHU ) %HFNHU -0
5HVLGXHV LQ WKH ¿UVW H[WUDFHOOXODU ORRS RI D * SURWHLQFRXSOHG
receptor play a role in signal transduction J Biol Chem 277(34),
'2, MEF0
/HH %. KDUH 6 1DLGHU ) %HFNHU -0 ,GHQWL¿FDWLRQ
of residues of the Saccharomyces cerevisiae G protein-coupled
receptor contributing to alpha-factor pheromone binding J
Biol Chem 276(41) '2, MEF
0
/LQ-&3DUULVK:(LOHUV06PLWK62 .RQRSND-%
Aromatic residues at the extracellular ends of transmembrane
domains 5 and 6 promote ligand activation of the G protein-coupled
alpha-factor receptor Biochemistry, 42(2) '2,
ELR
6RQ&'6DUJV\DQ+1DLGHU) %HFNHU-0,GHQWL¿FDWLRQRI
ligand binding regions of the Saccharomyces cerevisiae alpha-factor
SKHURPRQHUHFHSWRUE\SKRWRD൶QLW\FURVVOLQNLQJBiochemistry,
43(41) '2, EL
20 &KRL < RQRSND -% $FFHVVLELOLW\ RI F\VWHLQH UHVLGXHV
substituted into the cytoplasmic regions of the alpha-factor receptor
LGHQWL¿HVWKHLQWUDFHOOXODUUHVLGXHVWKDWDUHDYDLODEOHIRU*SURWHLQ
interaction Biochemestry 45(51) '2,
EL
21 6KL/ -DYLWFK-$7KHVHFRQGH[WUDFHOOXODUORRSRIWKHGRSDPLQH
D2 receptor lines the binding-site crevice Proc Natl Acad Sci
USA, 101(2) '2, SQDV
22 0D]QD3%HUND.-HOLQNRYD,%DOLN$6YRERGD32EVLORYD
92EVLO7 7HLVLQJHU-/LJDQGELQGLQJWRWKHKXPDQ07
PHODWRQLQUHFHSWRU WKHUROHRIUHVLGXHVLQWUDQVPHPEUDQHGRPDLQV
DQGBiochem Biophys Res Commun 332(3)
'2, MEEUF
23 .OHLQDX* .UDXVH*7K\URWURSLQDQGKRPRORJRXVJO\FRSURWHLQ
KRUPRQHUHFHSWRUV VWUXFWXUDODQGIXQFWLRQDODVSHFWVRIH[WUDFHOOXODU
signaling mechanisms Endocr Rev 30(2) '2,
HU
24 Unal, H., -DJDQQDWKDQ5%KDW0% .DUQLN66/LJDQG
VSHFL¿FFRQIRUPDWLRQRIH[WUDFHOOXODUORRSLQWKHDQJLRWHQVLQ
II type 1 receptor J Biol Chem 285(21)±
'2, MEF0
25 :LÀLQJ'%HUQKDUGW*'RYH6%XVFKDXHU$3HHWHUV0
:HVWHQ*YDQ/L4$3,:KHDWOH\0:RRWWHQ'&RQQHU
06LPPV-.HQGULFN50DVVRWWH'.LH൵HU%.OFR-
:LHJDQG&1DU]LQVNL.%DUDQVNL76XP&7LNKRQRYD,
&RVWDQ]L6*HUVKHQJRUQ01DQHYLF]7:DQJ/&KHQ0
,VKLL0&RXJKOLQ66FDUVHOOL0/L%.LP6:HVV-
%UXQVNROH,6WUDVVHU$6HLIHUW5%XVFKDXHU$:LÀLQJ'
/|൵HO.1RUGHPDQQ86WUDVVHU$%HUQKDUGW*'DYLGVRQ
)/RHZHQ3.KRUDQD+6KL/-DYLWFK-1RGD.6DDG
<*UDKDP5.DUQLN6&RRN-(LGQH.6KL/-DYLWFK
-$YODQL9 *UHJRU\ 0RUWRQ & 3DUNHU 0 6H[WRQ 3
/HINRZLW]5&RWHFFKLD66DPDPD3&RVWD70LOOLJDQ*
6HLIHUW5:HQ]HO6HLIHUW./HH7*HWKHU86DQGHUV%XVK
( 6FKQHOO ' %UXQVNROH , /DGRYD 6FKQHLGHU ( ,JHO
36HLIHUW56WUDVVHU$6FKQHLGHU(1HXPDQQ''RYH
6/LP+-RQJHMDQ$%DNNHU5+DDNVPD((VFK,GH
+DJD..UXVH$$VDGD+<XUXJL.RED\DVKL76KLURLVKL
05DVPXVVHQ6&KRL+)XQJ-3DUGRQ(&DVDURVD3
6KLPDPXUD7 6KLURLVKL0:H\DQG67VXMLPRWR+:LQWHU
G., Schneider, E., Schnell, D., Papa, D., Seifert, R., Igel, P., Geyer, R., Strasser, A., Dove, S., Seifert, R., Igel, P., Schneider, E., Schnell, '(O]66HLIHUW5/DQJH-:DOV+9DQGHQKRRJHQEDQG$ 9DQGHNXLOHQ$'HQKDUWRJ--DEORQRZVNL-*ULFH&&KDL: 'YRUDN&9HQDEOH-/LP+6PLWV5%DNNHU5'DP& YDQ(VFK,GH 6FKPXW]- .XHQ]OH*+XQ]LNHU)*DXFK5 6PLWV5/LP+6WHJLQN%%DNNHU5(VFK,GH*HWKHU8 /LQ6.RELOND%&KHQJ<3UXVR൵:6FKQHLGHU(6FKQHOO '6WUDVVHU$'RYH66HLIHUW5.RRLVWUD$.XKQH6(VFK ,GH/HXUV5*UDDI&GH/LP+*UDDI&GH-LDQJ: 6DGHN30F*RYHUQ36FKXOWHV61LMPHLMHU6(QJHOKDUGW+ RRLVWUD$9LVFKHU+:LWWPDQQ+-6HLIHUW56WUDVVHU$
$OHZLMQVH$7LPPHUPDQ+-DFREV(6PLW05RRYHUV(
%DOOHVWHURV- :HLQVWHLQ+7KH([WUDFHOOXODU/RRS (&/
of the Human Histamine H 4 Receptor Substantially Contributes
WR /LJDQG %LQGLQJ DQG &RQVWLWXWLYH$FWLYLW\ PloS One, 10(1),
H '2, MRXUQDOSRQH
26 <DQJ-<DQ55R\$;X'3RLVVRQ- =KDQJ<7KH
,7$66(56XLWH SURWHLQVWUXFWXUHDQGIXQFWLRQSUHGLFWLRQNature
Methods, 12(1) '2, QPHWK
(LOHUV0+RUQDN96PLWK62 .RQRSND-%&RPSDULVRQ RI&ODVV$DQG'*3URWHLQ&RXSOHG5HFHSWRUV &RPPRQ)HDWXUHV
in Structure and Activation Biochemistry 2005, 44
'2, ELX
3DOF]HZVNL..XPDVDND7+RUL7%HKQNH&$0RWRVKLPD H., Fox, B.A., /H7URQJ,7HOOHU'&2NDGD76WHQNDPS 5(<DPDPRWR0 0L\DQR0&U\VWDOVWUXFWXUHRIUKRGRSVLQ
AG protein-coupled receptor Science, 289(5480)
%HUPDQ +0:HVWEURRN - )HQJ = *LOOLODQG * %KDW7 1:HLVVLJ+6KLQG\DORY,1 %RXUQH3(7KH3URWHLQ'DWD
Bank Nucleic Acids Res 28(1) '2,
QDU
30 0DXSHWLW-'HUUHXPDX[3 7X൵pU\3$IDVWPHWKRGIRUODUJH
scale de novo peptide and miniprotein structure prediction J
Comput Chem 31(4) '2, MFF
31 +LJDVKLMLPD70DVXL<&KLQR16DNDNLEDUD6.LWD+ 0L\D]DZD 7 1XFOHDUPDJQHWLFUHVRQDQFH VWXGLHV RQ WKH conformations of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution
Conformation-activity relationship Eur J Biochem 140(1),
±
32 &RPHDX65*DWFKHOO':9DMGD6 &DPDFKR&-&OXV3UR
an automated docking and discrimination method for the prediction
of protein complexes Bioinformatics, 20(1), 45-50 (2004)
33 1DLGHU)%HFNHU-0/HH<+ +RURYLW]$'RXEOHPXWDQW cycle scanning of the interaction of a peptide ligand and its G
protein-coupled receptor Biochemistry, 46(11)
'2, ELX
34 8PDQDK*.(+XDQJ/'LQJ)$UVKDYD%)DUOH\$5 /LQN$-1DLGHU) %HFNHU-0,GHQWL¿FDWLRQRIUHVLGXHWR UHVLGXHFRQWDFWEHWZHHQDSHSWLGHOLJDQGDQGLWV*SURWHLQFRXSOHG receptor using periodate-mediated dihydroxyphenylalanine
cross-linking and mass spectrometry J Biol Chem., 285(50)
'2, MEF0
35 %URRNV %5 %UXFFROHUL 5( 2ODIVRQ %' 6WDWHV '- 6ZDPLQDWKDQ 6 DUSOXV 0 &+$500 $ SURJUDP IRU macromolecular energy, minimization, and dynamics calculations
Journal of Computational Chemistry, 4(2) '2,
MFF
... UHVLGXHplaced at the end of a helix and the alpha- carbon from the
residues localized at the ends of all the other helices
Construction of the alpha- pheromone three-dimensional...
SUHYLRXVO\)RUH[DPSOH3KHGRHVQRWLQWHUDFWZLWK7\U
of the alpha pheromone in the Umanah model34 The lack of
LQWHUDFWLRQ EHWZHHQ WKHVH UHVLGXHV ZDV PRGHOHG EDVHG RQ
studies of alpha pheromone analogues... residues of Ste2p and pheromone are shown in a ball-and-stick representation First, the C-terminal region of the pheromone (Gln10) binds to the receptor residues Ser47 and Thr48 (H1) 17 Then, the