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Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2 -containing membranes

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The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their
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                      George Khelashvili 1 , Milka Doktorova 1 , Michelle A. Sahai 1 ,  Niklaus Johner  2 , Lei Shi 1,3 , and arel !einstein 1,3 1  !eill "ornell Medical "ollege o# "ornell $niversit%, De&art'ent o# (h%siolog% and )io&h%sics, Ne* +ork, N+, 1-, $SA 2 $niversit% o# )asel, )io/entru' 0 "enter #or Molecular Sciences, Klingelergstrasse , "04- )asel, S*it/erland 3  !eill "ornell Medical "ollege o# "ornell $niversit%, 5 (rince Al*aleed )in 6alal )in Adula/i/ Alsaud 7nstitute o# "o'&utational )io'edicine, Ne* +ork, N+, 121, $SA "orres&onding Author8    Address 8 13 +ork Avenue, 5oo' L"01A, Ne* +ork, N+, 1-  Email  8 gek29:'ed.cornell.edu;  Phone 8 21204-0-3<-;  Fax 8 21204-0-22-   Modeling o#    DA6 N0ter'inus(7( 2  interactions !   Neurotrans'itter trans&orter, sodiu' s%'&orter, signaling, e##lu=, a'&heta'ine, &hos&hor%lation, 'olecular d%na'ics, electrostatics Research ArticleProteins: Structure, Function and BioinformaticsDOI 10.1002/prot.24792This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/prot.24792© 2015 Wiley Periodicals, Inc.Received: Dec 16, 2014; Revised: Feb 05, 2015; Accepted: Feb 24, 2015 This article is protected by copyright. All rights reserved.  2 " 6he do&a'ine trans&orter >DA6? is a trans'e'rane &rotein elonging to the #a'il% o#  Neurotrans'itter8Sodiu' S%'&orters >NSS?. Me'ers o# the NSS are res&onsile #or the clearance o# neurotrans'itters #ro' the s%na&tic cle#t, and #or their translocation ack into the  &res%na&tic nerve ter'inal. 6he DA6 contains long intracellular N0 and "0ter'inal do'ains that are strongl% i'&licated in the trans&orter #unction. 6he N0ter'inus >N0ter'?, in  &articular, regulates the reverse trans&ort >e##lu=? o# the sustrate through DA6. "urrentl%, the 'olecular 'echanis's o# the e##lu= re'ain elusive in large &art due to lack o# structural in#or'ation on the N0ter'inal seg'ent. ere *e re&ort a co'&utational 'odel o# the N0ter' o# the hu'an DA6 >hDA6?, otained through an ab initio  structure &rediction, in co'ination *ith e=tensive ato'istic 'olecular d%na'ics >MD? si'ulations in the conte=t o# a li&id 'e'rane. @ur anal%sis reveals that *hereas the N0ter' is a highl% d%na'ic do'ain, it contains secondar% structure ele'ents that re'ain stale in the long MD traectories o# interactions *ith the ila%er >totaling B2.2 Cs?. "o'ining MD si'ulations *ith continuu' 'ean0#ield 'odeling *e #ound that the N0ter' engages *ith li&id 'e'ranes through electrostatic interactions *ith the charged li&ids (7( 2  >&hos&hatid%linositol 4,0)i&hos&hate? or (S >&hos&hatid%lserine? that are &resent in these ila%ers. !e identi#% s&eci#ic 'oti#s along the N0ter' i'&licated in such interactions and sho* that di##erential 'odes o# N0ter''e'rane association result in di##erential &ositioning o# the structured seg'ents on the 'e'rane sur#ace. 6hese results *ill in#or' #uture structure0ased studies that *ill elucidate the 'echanistic role o# the N0ter' in DA6 #unction.  Page 2 of 46John Wiley & Sons, Inc.PROTEINS: Structure, Function, and Bioinformatics This article is protected by copyright. All rights reserved.  3 6he do&a'ine trans&orter >DA6? is a 'e'rane &rotein in the #a'il% o# 'a''alian  Neurotrans'itter8Sodiu' S%'&orters >NSS? that also includes the closel% related trans&orters #or serotonin >S56? and nore&ine&hrine >N6? 103 . 6he NSS are res&onsile #or the clearance o# neurotrans'itters >e.g. do&a'ine >DA?, % DA6? #ro' the s%na&tic cle#t, and their translocation ack into the c%to&las' o# the &res%na&tic neuronal cell. 6he u&take o# neurotrans'itter is 'ade &ossile % cou&ling the trans'e'rane >6M? Na E gradient to the u&hill trans&ort o# the res&ective sustrate 2 . 6he #unctions o# NSS &roteins in neuronal signaling i'&licate the' in a nu'er o# &s%chiatric and neurological disorders that include schi/o&hrenia, and (arkinsonFs disease 4 , and in the 'echanis's o# action o# aused  &s%chosti'ulants, such as cocaine and a'&heta'ine >AM(? ,- . 6heir essential neuro&h%siological roles have 'ade these trans&orters &ri'ar% targets #or antide&ressant 'edications. 6he #irst 0ra% structure o# the NSS DA6 #ro'  Drosophila  >dDA6? *as re&orted recentl%  , and revealed 12 6M seg'ents, a do'ain architecture that has een &redicted as *ell #ro' &revious 'olecular 'odeling o# hu'an DA6 >hDA6? ased on seHuence ho'olog% to the acterial Leucine trans&orter >Leu6? <014  #or *hich several cr%stal structures have e=isted since 2 1022 . )ut in contrast to Leu6, DA6 has 'uch longer c%to&las'ic N0 and "0ter'inal seg'ents. 6hese intracellular seg'ents &ossess nu'erous &utative &hos&hor%lation sites, and several &rotein kinases have een i'&licated in the regulation o# DA6 #unction 2302- . 6he &hos&hor%lation o# the N0ter'inal seg'ent at serine residues &ositioned in its distal  &ortions >i.e., close to its starting residue, Met1, see Iigure 1A?, leads to the intriguing  &henot%&e o# e##lu= in *hich the sustrate, DA, is trans&orted via DA6 in the reverse direction, out o# the cell 2031 . $nder &h%siological conditions, e##lu= can e triggered % the action o# the &s%chosti'ulant a'&heta'ine >AM(?, *hich a&&arentl% leads to a DA6 con#or'ation suitale #or the &hos&hor%lation o# the N0ter'inus. 7ndeed, in studies o# Page 3 of 46John Wiley & Sons, Inc.PROTEINS: Structure, Function, and Bioinformatics This article is protected by copyright. All rights reserved.  4  &hos&ho'i'etic S0to0D 'utations sustituting the distal serine residues o# the N0ter'inus >N0ter'?, DA60'ediated DA e##lu= *as oserved even in the asence o# AM( 2 . 7nterestingl%, e##lu= can e regulated se&aratel% #ro' the sustrate u&take &rocess. 6hus, *e have sho*n that #or hDA6, charge0neutrali/ing sustitution o# K3 and K residues in the  N0ter' *ith either Ala or Asn dra'aticall% reduces AM(0induced DA e##lu= *hile leaving the DA u&take unchanged 32 . 6he sa'e studies have suggested a central 'echanistic role #or direct inding o# the N0ter' to highl% charged anionic (7( 2  >&hos&hatid%linositol 4,0)i&hos&hate? li&ids in the e##lu= &rocess 32 . 6he i'&ortance o# the e##lu= &rocess is underscored % results #ro' recent studies sho*ing that it is a##ected % s&eci#ic de novo  'utations in DA6 linked to various neurological disorders 3303< . Des&ite ra&id &rogress in identi#%ing ke% ele'ents o# the 'olecular 'achiner% that regulates the e##lu= &rocess in NSS &roteins >e.g., involve'ent o# s&eci#ic co'&onents o# the cell 'e'rane, interactions *ith sca##olding &roteins? 39042 , the 'olecular 'echanis' o# DA60'ediated reverse trans&ort re'ains unclear. 6his includes 'echanistic Huestions aout the role o# the N0ter'inal seg'ent >residues 109 in hDA6?, #or *hich structural in#or'ation is lacking ecause this #unctionall% i'&ortant region in DA6 is asent in the &rotot%&ical  NSS, the acterial trans&orter Leu6, and also had to e e=cised #ro' the construct used to otain the onl% availale 0ra% structure o# the DA6 &rotein #ro'  Drosophila  >dDA6?  . Moreover, the seHuence o# the DA6 N0ter'inus is not ho'ologous to an% &rotein *ith kno*n #old, and e=hiits i'&ortant variations a'ong DA6 &roteins #ro' di##erent s&ecies >Iigure 1A?. 6o overco'e this di##icult%, *e sought a &rediction o# the three0di'ensional >3D? con#or'ation o# the N0ter'inus #ro' the hu'an DA6 >hDA6? % co'ining, as descried here, ab initio  structure &rediction tools and e=tensive ato'istic 'olecular d%na'ics >MD? si'ulations. 6he ab initio 'odeling, carried out *ith the 5osetta so#t*are 43 , %ielded Page 4 of 46John Wiley & Sons, Inc.PROTEINS: Structure, Function, and Bioinformatics This article is protected by copyright. All rights reserved.  5  &redictions o# structured regions *ithin the #irst  residues o# the N0ter'. SuseHuent long >B2.2 Cs in total? ato'istic MD si'ulations o# the N0ter' in co'&le= *ith a li&id 'e'rane sho*ed that the identi#ied secondar% structure ele'ents *ere stale on the si'ulation ti'escales, and *ere involved in s&eci#ic interactions *ith &ertinent 'odels o# the cell 'e'rane. Notal%, *e #ound that the s&atial orientation and &osition relative to the 'e'rane o# these structured ele'ents is largel% deter'ined % s&eci#ic 'odes o# N0ter''e'rane association. Anal%sis o# the MD traectories and evaluation o# 'e'rane res&onses to interactions *ith the N0ter', using Huantitative sel#0consistent 'ean0#ield 'odeling 4404 , sho*ed that the N0ter' engages in strong electrostatic interactions *ith the li&id ila%er through contacts et*een several asic residues and charged (7( 2  or (S >&hos&hatid%lserine? li&ids. Notal%, *hen the &redicted N0ter'inus 'odel *as attached to the #ull 6M undle o# the cr%stallogra&hicall% solved dDA6 structure >sustituting #or the inco'&lete dDA6 ter'inus?, the MD si'ulations >- ns in total? revealed si'ilar 'odes o# interactions *ith the li&id 'e'rane. 6hese constructs, and the 'olecular insights regarding the &re#erred 'odes o# interaction o# the DA6 N0ter' *ith 'odel 'e'ranes otained #ro' the stud%, &rovide a novel structural conte=t #or #uture e=&lorations o# the #unctional 'echanis's o# this NSS0#a'il% trans&orter. #           Page 5 of 46John Wiley & Sons, Inc.PROTEINS: Structure, Function, and Bioinformatics This article is protected by copyright. All rights reserved.
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