One of the essential structures required by every living organism is a membrane that encompasses the cytoplasm (the contents of the cell). Membranes, not only have a very important role in sustaining contacts with the outside world, but also in protecting the cell against it. Whilst membranes have to be quite resistant to the stressful environment surrounding the cell, they also have to enable uptake of substrates and extrusion of waste products. .... 

Zie: Summary

Sobczak, Iwona,

Sobczak - Elbourne, Iwona

ARTS repository - University of Groningen

   University of GroningenStructural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniaeSobczak - Elbourne, IwonaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Sobczak - Elbourne, I. (2005). Structural and functional features of the Na+-citrate transporter CitS ofKlebsiella pneumoniae. [Thesis fully internal (DIV), University of Groningen]. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-09-2023VIISummarvOne of thc essential structures rcquired by er"ery l iving organisnr is a rncmbranc thatencompasses the cytoplasm (the contents of the cel l) .  Membranes. not only have a veryimportant role in sustaining contacts with the outside rvorld, but also in protcct ing the cel lagainst i t .  Whilst membranes have to be quite resistant to the stressfl l  enyironmentsurrounding the cel l ,  thcy also have to enable uplake of substrates and extrusion of g'asteproducts. In other words only certain compounds are al lon'ed to pernteate through thatbarrier.Plasma rnembranes are composed of two rnain components: l ipids and proteins, the lattcrplay a very inlportant role in contact with the environment. Some rnembrane proteins areresponsible Íbrthe uptake of required compounds. There are two types of these proteins thatpart icipate in the transport of solutes and ions across the membrane. The l lrsr rype are poresand channels that làci l i tate the dif l i rsion of substrates without rcquir ing metabolic energy(Fig. lA). Thcy Íbrm structt lres that resernblc "holes" in the mcrnbrane. The second type arernembrane transportcrs: proteins that show a great varicty of transport rnechanisms,structures and specilicities. They are divided into 3 classes based on the energy required Íorthe transport process: ( i)  primary transporters (Fig. lB) use chemical cnergy released, i trrexample, in thc breakdown of ATP, ( i i )  secondary transpofters (Fig. lc) use cnergy storedin the electrochemical gradient of solutes and/or ions, ( i i i )  group translocators (Fig. lD)rnovc the solule and perfbrm its chernical rnodi l icat ion; the modiÍ led solute is released onthe clther side o1- the membrane. Mernbrane transpoftcrs interact with their substrates anclundergo structural changcs during the translocation process.The CitS protein is a sodiurn dcpendent citrate transporler lound in the pathogenic bacteriumKlebsiella pneunoniue. It belongs to the class ofsecondary transporters. It transports citratetogcther n' i th 2 sodium ions in the same direct ion ( into the bacterial cel l)  (Fig. lc).Therefore, it is termed a symporter, u,hich diÍïèrs liom the other types of secondarytransporters that eithcr movc substratcs in opposite directions (antiportcrs) or transpod onlyonc substrate independently of any coupling ion in one dircct ion (uniporter).  The transportolci trate is driven by the electrochernical gradient of the sodium ions across the nrembrane.This is knor.vn as the sodiurn ion motive Íbrce (smÍ).The CitS protein is very specif ic because i t  only recognises the citrate anion (citr-) as aslrbstratc. This suggests that thc structure ofthc protein has to be rvel l  aclapted to the role ofChapter  Vl lrcco-{nit ion, binding and translocation ol only this substrate. Studies of theÍirnction of thc transport proteins try to understand these processes takingrnolccular lcvcl and aim. ult imatcly, to cxplain the mechanisrn of transport.s1tï'ffi+lS , istructlrre andplace at theC-'hapter 4 of thydrophobictowards the c'Amino acid r,easi ly reachecthat the residuthe residues almemoFigure 2. l\(.Ía/a'r ) betwtornls a porrtrallslocatl0rVb corrcsprinver ted  or if  y  top lasmi rThc cytoplasnPart of that loprotein Íamil lStudies of th,shou ed that I(outside) sideÍiom thc perip(Chapter 3) reloop that ÍoldDcBA5S,Àb\^:I n  b - rATP )z,.r,rJ,.l-ï#r .,.,.:;:'-,r:ièr i di :--f pyruvate, - DIPEPl ' i gurc  l  D i Í Í c ren t  t rpes  o f t ranspor t  across  the  membrane:  (A)  channe l .  (B)  p r i r ra ry  t ranspor tc r .  (C)sccondarr transporter. (D) group translocator. Symbols and abbreviations: S substrate of thetransporter. A I 'P adcnosine 5'-triphosphate, ADP adenosine diphosphate. Pi Í iee phosphate group,P[P phosphocno lopyruvate ,  S-P phosphory , la tec l  sugar .  l lA .  l lB  and I IC  th ree  subun i ts  o Í '1he  sanrcpro te in ,  HPr  and EI  o thcr  cnz lmcs in lo l led  in  the  t rans lè r  o fphosphory l  g roup.Erery protein is bui l t  Í iom chains o1-amino acids of dif lerent lengths, cal led polypeptidechains. Such polypeptide chains Íbld into three-dinrensional structures, which give theproteins their specif ic functions. I lecause membrane proteins insert themselves into therrembrane rn a zigzag Íashion, part of the polypeptide chain fbnns the characterist ic hel icalstructure inside ol-the hydrophobic membrane environment.CitS consists of a 446 amino acids long polypeptide chain that traverses the membrane 11times and thus lbrms 1 I rnembrane-embedded parts (Fig. 2). These parts, namedlransmembrane segments (TMSs), Íbrm the hel ical structure. In contrast, the loops aremostly unstructr.rred and are local ised in the cytoplasm (inside) orperiplasm (outside) of the(Ciram-negative) bactcrial cel l .  In CitS there are a Íbu, large cytoplasmic loops, rvi th theIonsest onc connccting the TMS VI and VII (Fig. 2). The loop bctwccn TMS VIII  and IX(AH; Fig.2) is predicted by computational methods to also l i rrrn a hel ical structure. In134'ucture andlace at the)yruvate'ner. (C )r  of  the:e group.the samcrolypeptideh give thees into thestic hel ical:mbrane I IÍs, namedr loops areside) of thes, rvith the/ l l l  and  IX.ructure. InSummaryChapter 4 of this thcsis, i t  is shown that this loop is f i r lded as an arnphipathic hel ix, with thehydrophobic Íacc of the hel ix exposed tor 'vards the membranc and the hydrophil ic Íacetou'ards thc cytoplasm. This amphipathic Íèature was the key to the experirnental evidence.Amino acid residues at the hydrophobic Íàce of thc hel ix, close to the membrane, are noteasily reached by the u'ater-soluble reagcnts uscd to rnodily the residues. We havc shorvnthat the residues located on thc hydrophobic Íàce are not accessible Íbr such reagents, rvhi lethe residues at the hydrophil ic Íace arc.membraneFigure  2 .  Membrane topo log l  mode l  o f  the  C i ts  p ro te in .  TMSs are  ind ica ted  by  bores .  The loop AH(gre,t) betu'een TMS Vll l and IX tbrms an amphiprthic hclir. Loop Xa, the best-conserved region,lbrms a pore-loop stnlcturc and logclher s' ith TMS X and XI (t lu.shed bores\ is involved in thc substratetranslocation path['a1'. Trvo clsteine residues in the Xa region are represented as hlutk cirt lc.s. ScgmentVb corresponds kr a h1'drophobic rcgion: i l ' the protcin consists ol ' trvo homologous dorrains r,ithin \e r ted  o Í ien ta t ion .  each o i Í i ve  TMSs ( ind ica tcd  as  lushet l  àores) ,  Vb poss ib ly  fomrs  a  pore- loop.C l top lasnr ic  ( i i )  anc l  per ip lasmic  (oa l )  s ides  o f the  membranc  are  ind ica tcd .The cytoplasmic loop locatcd bctr.r,een TMS X and XI showed another unexpected structure.Part o1-that loop, cal led Xa, comprises the least changed region in the evolut ion of the CitSprotein Íàmily; i t  rvas postulatcd to be inr. 'olved in the binding of the substrate (Fig. 2).Studies of the trvo cysteine residues located in the cytoplasmic Xa region (Chapter 2)sho*'ed that they can be accessed by small  impermeable reagcnts from the periplasmic(outside) side of the membrane. Ho\\ 'evcr, largcrreagents were not able to access this regionÍiom the periplasm indicating that thcre is sorne size-restr icted cavity. More detai led studics(Chapter 3) revealed that this region Íonns a structure cal led a pore-loop, which is parl  of aloop that Íblds back into thc mcmbranc between the TMSs but never protrudes completcly1 3 5Chapter Vllto thc other side. This cxplains why undcr sortre condit ions. certain residues are easi lyaccessible Í iorn the other side of the n-rcmbrane. Pore-loop struclures are common inchannels but not in secondary transporters. Only in one làmily of secondary transporters, theglutanrate transp(n' lers, har. 'e pore-loops been clbsen ed in rcgions closely involr ed insuhstrate binding. l t  is thought that they lbnn a pathway Íbr the substrate and, by theirllexible structure, inducc and control thc changes in thc structure of the prcÍein aÍ1er thesubstrate is bound. As shou'n lbr ( ' i tS in Chapter I  of this thesis, binding of ci trate could, tosonre e\tent, inhibit  the accessibi l i t ) '  of the probcd residues. Surprisingly, complcteinhibit ion u'as achier"cd only in the prcscnce ot '  sodir-rm ions. Since sodiunr iorrs lre torrsmall  to causc pÍotection against the reagent by covering thc access to the rcsidues, astructural change in the protcin is probably thc main reason Íbr the observed effcct.One l irr ther interestin-u paft t l l - thc plotein is the large periplasrnic rcgion cal led Vb. The Vbsegrnent had bccn preriously prcdicted to lbrm a transmembranc segrnent lr l l t  cxpcrimentsshou'ed that i t  is exported into the periplasnr. A fecent analysis ol-the amino acid sequenceof thc secondary transpoftcrs'  prol i les in the CitS Íarni ly suggested the presence of trvodornir ins in thc protein structurc. For CitS, i t  is proposed (Chapter 1) that thc clonrainsconsist of Í lve TMSs and that TMS I is an addit ional scgnrent. According to the dornainnrrrdel,  region Xa is hornologous to rcgion Vb sLrggcsting that Vb could Íbrm another pore-lonp structure and that the two domajns have an inverted orientat ion in the rnembrane (Fig.a \Thc main object of thc research described in this thesis nas to obtain more inl irrrnationabor.rt  the structure ol ' thc CitS protein and i ts rclat ion to the function. The presence of theporc-loop in the structure is not accidental and therelbre lvc have tr ied to cxplain the role ofthis region in the translocation ol-the substrate. Besidcs, the role of thc arnphipathic hcl irAl l  and legions l ike Vb in the protein nrust be erplained. I t  is possible that such structuresare more corrnlon in transport protcins but rather diÍ ï lcult  to prcdict and local ise. Moreinlormation on membrane protein structurcs and transport mechanisms wil l  be helpful in thcÍuturc tbr a better understandin.e of the lunction and importance of these proteins Íor l iv ingorganrsrrs.Een essentiëlinhoud vanonderlroudenbuitenu e reldÍroeten z€ 0,Met anderedoorbreken.Plasmamenitspelen een btzijn verantrvtdezc eiwitterccrste tyllemetabole cnmembraan. lvertonen invr--rdeeld op I( i)  prirnairebi jvoorbeeldener_sle opgetranslocaticrtn'orden. Tratl  erandering rHct Cl i tS eiubacterie K1c.transportecrte i rv i t  to t  de 'dic hun subsonaÍhankelr j lVan cltraat wmernbraan. [--136

English

Structural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniae

Proceedings - University of Groningen

   University of GroningenStructural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniaeSobczak - Elbourne, IwonaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Sobczak - Elbourne, I. (2005). Structural and functional features of the Na+-citrate transporter CitS ofKlebsiella pneumoniae. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 11-10-2022VIISummarvOne of thc essential structures rcquired by er"ery l iving organisnr is a rncmbranc thatencompasses the cytoplasm (the contents of the cel l) .  Membranes. not only have a veryimportant role in sustaining contacts with the outside rvorld, but also in protcct ing the cel lagainst i t .  Whilst membranes have to be quite resistant to the stressfl l  enyironmentsurrounding the cel l ,  thcy also have to enable uplake of substrates and extrusion of g'asteproducts. In other words only certain compounds are al lon'ed to pernteate through thatbarrier.Plasma rnembranes are composed of two rnain components: l ipids and proteins, the lattcrplay a very inlportant role in contact with the environment. Some rnembrane proteins areresponsible Íbrthe uptake of required compounds. There are two types of these proteins thatpart icipate in the transport of solutes and ions across the membrane. The l lrsr rype are poresand channels that làci l i tate the dif l i rsion of substrates without rcquir ing metabolic energy(Fig. lA). Thcy Íbrm structt lres that resernblc "holes" in the mcrnbrane. The second type arernembrane transportcrs: proteins that show a great varicty of transport rnechanisms,structures and specilicities. They are divided into 3 classes based on the energy required Íorthe transport process: ( i)  primary transporters (Fig. lB) use chemical cnergy released, i trrexample, in thc breakdown of ATP, ( i i )  secondary transpofters (Fig. lc) use cnergy storedin the electrochemical gradient of solutes and/or ions, ( i i i )  group translocators (Fig. lD)rnovc the solule and perfbrm its chernical rnodi l icat ion; the modiÍ led solute is released onthe clther side o1- the membrane. Mernbrane transpoftcrs interact with their substrates anclundergo structural changcs during the translocation process.The CitS protein is a sodiurn dcpendent citrate transporler lound in the pathogenic bacteriumKlebsiella pneunoniue. It belongs to the class ofsecondary transporters. It transports citratetogcther n' i th 2 sodium ions in the same direct ion ( into the bacterial cel l)  (Fig. lc).Therefore, it is termed a symporter, u,hich diÍïèrs liom the other types of secondarytransporters that eithcr movc substratcs in opposite directions (antiportcrs) or transpod onlyonc substrate independently of any coupling ion in one dircct ion (uniporter).  The transportolci trate is driven by the electrochernical gradient of the sodium ions across the nrembrane.This is knor.vn as the sodiurn ion motive Íbrce (smÍ).The CitS protein is very specif ic because i t  only recognises the citrate anion (citr-) as aslrbstratc. This suggests that thc structure ofthc protein has to be rvel l  aclapted to the role ofChapter  Vl lrcco-{nit ion, binding and translocation ol only this substrate. Studies of theÍirnction of thc transport proteins try to understand these processes takingrnolccular lcvcl and aim. ult imatcly, to cxplain the mechanisrn of transport.s1tï'ffi+lS , istructlrre andplace at theC-'hapter 4 of thydrophobictowards the c'Amino acid r,easi ly reachecthat the residuthe residues almemoFigure 2. l\(.Ía/a'r ) betwtornls a porrtrallslocatl0rVb corrcsprinver ted  or if  y  top lasmi rThc cytoplasnPart of that loprotein Íamil lStudies of th,shou ed that I(outside) sideÍiom thc perip(Chapter 3) reloop that ÍoldDcBA5S,Àb\^:I n  b - rATP )z,.r,rJ,.l-ï#r .,.,.:;:'-,r:ièr i di :--f pyruvate, - DIPEPl ' i gurc  l  D i Í Í c ren t  t rpes  o f t ranspor t  across  the  membrane:  (A)  channe l .  (B)  p r i r ra ry  t ranspor tc r .  (C)sccondarr transporter. (D) group translocator. Symbols and abbreviations: S substrate of thetransporter. A I 'P adcnosine 5'-triphosphate, ADP adenosine diphosphate. Pi Í iee phosphate group,P[P phosphocno lopyruvate ,  S-P phosphory , la tec l  sugar .  l lA .  l lB  and I IC  th ree  subun i ts  o Í '1he  sanrcpro te in ,  HPr  and EI  o thcr  cnz lmcs in lo l led  in  the  t rans lè r  o fphosphory l  g roup.Erery protein is bui l t  Í iom chains o1-amino acids of dif lerent lengths, cal led polypeptidechains. Such polypeptide chains Íbld into three-dinrensional structures, which give theproteins their specif ic functions. I lecause membrane proteins insert themselves into therrembrane rn a zigzag Íashion, part of the polypeptide chain fbnns the characterist ic hel icalstructure inside ol-the hydrophobic membrane environment.CitS consists of a 446 amino acids long polypeptide chain that traverses the membrane 11times and thus lbrms 1 I rnembrane-embedded parts (Fig. 2). These parts, namedlransmembrane segments (TMSs), Íbrm the hel ical structure. In contrast, the loops aremostly unstructr.rred and are local ised in the cytoplasm (inside) orperiplasm (outside) of the(Ciram-negative) bactcrial cel l .  In CitS there are a Íbu, large cytoplasmic loops, rvi th theIonsest onc connccting the TMS VI and VII (Fig. 2). The loop bctwccn TMS VIII  and IX(AH; Fig.2) is predicted by computational methods to also l i rrrn a hel ical structure. In134'ucture andlace at the)yruvate'ner. (C )r  of  the:e group.the samcrolypeptideh give thees into thestic hel ical:mbrane I IÍs, namedr loops areside) of thes, rvith the/ l l l  and  IX.ructure. InSummaryChapter 4 of this thcsis, i t  is shown that this loop is f i r lded as an arnphipathic hel ix, with thehydrophobic Íacc of the hel ix exposed tor 'vards the membranc and the hydrophil ic Íacetou'ards thc cytoplasm. This amphipathic Íèature was the key to the experirnental evidence.Amino acid residues at the hydrophobic Íàce of thc hel ix, close to the membrane, are noteasily reached by the u'ater-soluble reagcnts uscd to rnodily the residues. We havc shorvnthat the residues located on thc hydrophobic Íàce are not accessible Íbr such reagents, rvhi lethe residues at the hydrophil ic Íace arc.membraneFigure  2 .  Membrane topo log l  mode l  o f  the  C i ts  p ro te in .  TMSs are  ind ica ted  by  bores .  The loop AH(gre,t) betu'een TMS Vll l and IX tbrms an amphiprthic hclir. Loop Xa, the best-conserved region,lbrms a pore-loop stnlcturc and logclher s' ith TMS X and XI (t lu.shed bores\ is involved in thc substratetranslocation path['a1'. Trvo clsteine residues in the Xa region are represented as hlutk cirt lc.s. ScgmentVb corresponds kr a h1'drophobic rcgion: i l ' the protcin consists ol ' trvo homologous dorrains r,ithin \e r ted  o Í ien ta t ion .  each o i Í i ve  TMSs ( ind ica tcd  as  lushet l  àores) ,  Vb poss ib ly  fomrs  a  pore- loop.C l top lasnr ic  ( i i )  anc l  per ip lasmic  (oa l )  s ides  o f the  membranc  are  ind ica tcd .The cytoplasmic loop locatcd bctr.r,een TMS X and XI showed another unexpected structure.Part o1-that loop, cal led Xa, comprises the least changed region in the evolut ion of the CitSprotein Íàmily; i t  rvas postulatcd to be inr. 'olved in the binding of the substrate (Fig. 2).Studies of the trvo cysteine residues located in the cytoplasmic Xa region (Chapter 2)sho*'ed that they can be accessed by small  impermeable reagcnts from the periplasmic(outside) side of the membrane. Ho\\ 'evcr, largcrreagents were not able to access this regionÍiom the periplasm indicating that thcre is sorne size-restr icted cavity. More detai led studics(Chapter 3) revealed that this region Íonns a structure cal led a pore-loop, which is parl  of aloop that Íblds back into thc mcmbranc between the TMSs but never protrudes completcly1 3 5Chapter Vllto thc other side. This cxplains why undcr sortre condit ions. certain residues are easi lyaccessible Í iorn the other side of the n-rcmbrane. Pore-loop struclures are common inchannels but not in secondary transporters. Only in one làmily of secondary transporters, theglutanrate transp(n' lers, har. 'e pore-loops been clbsen ed in rcgions closely involr ed insuhstrate binding. l t  is thought that they lbnn a pathway Íbr the substrate and, by theirllexible structure, inducc and control thc changes in thc structure of the prcÍein aÍ1er thesubstrate is bound. As shou'n lbr ( ' i tS in Chapter I  of this thesis, binding of ci trate could, tosonre e\tent, inhibit  the accessibi l i t ) '  of the probcd residues. Surprisingly, complcteinhibit ion u'as achier"cd only in the prcscnce ot '  sodir-rm ions. Since sodiunr iorrs lre torrsmall  to causc pÍotection against the reagent by covering thc access to the rcsidues, astructural change in the protcin is probably thc main reason Íbr the observed effcct.One l irr ther interestin-u paft t l l - thc plotein is the large periplasrnic rcgion cal led Vb. The Vbsegrnent had bccn preriously prcdicted to lbrm a transmembranc segrnent lr l l t  cxpcrimentsshou'ed that i t  is exported into the periplasnr. A fecent analysis ol-the amino acid sequenceof thc secondary transpoftcrs'  prol i les in the CitS Íarni ly suggested the presence of trvodornir ins in thc protein structurc. For CitS, i t  is proposed (Chapter 1) that thc clonrainsconsist of Í lve TMSs and that TMS I is an addit ional scgnrent. According to the dornainnrrrdel,  region Xa is hornologous to rcgion Vb sLrggcsting that Vb could Íbrm another pore-lonp structure and that the two domajns have an inverted orientat ion in the rnembrane (Fig.a \Thc main object of thc research described in this thesis nas to obtain more inl irrrnationabor.rt  the structure ol ' thc CitS protein and i ts rclat ion to the function. The presence of theporc-loop in the structure is not accidental and therelbre lvc have tr ied to cxplain the role ofthis region in the translocation ol-the substrate. Besidcs, the role of thc arnphipathic hcl irAl l  and legions l ike Vb in the protein nrust be erplained. I t  is possible that such structuresare more corrnlon in transport protcins but rather diÍ ï lcult  to prcdict and local ise. Moreinlormation on membrane protein structurcs and transport mechanisms wil l  be helpful in thcÍuturc tbr a better understandin.e of the lunction and importance of these proteins Íor l iv ingorganrsrrs.Een essentiëlinhoud vanonderlroudenbuitenu e reldÍroeten z€ 0,Met anderedoorbreken.Plasmamenitspelen een btzijn verantrvtdezc eiwitterccrste tyllemetabole cnmembraan. lvertonen invr--rdeeld op I( i)  prirnairebi jvoorbeeldener_sle opgetranslocaticrtn'orden. Tratl  erandering rHct Cl i tS eiubacterie K1c.transportecrte i rv i t  to t  de 'dic hun subsonaÍhankelr j lVan cltraat wmernbraan. [--136

University of Groningen Digital Archive

   University of GroningenStructural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniaeSobczak - Elbourne, IwonaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Sobczak - Elbourne, I. (2005). Structural and functional features of the Na+-citrate transporter CitS ofKlebsiella pneumoniae. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 07-06-2022VIISummarvOne of thc essential structures rcquired by er"ery l iving organisnr is a rncmbranc thatencompasses the cytoplasm (the contents of the cel l) .  Membranes. not only have a veryimportant role in sustaining contacts with the outside rvorld, but also in protcct ing the cel lagainst i t .  Whilst membranes have to be quite resistant to the stressfl l  enyironmentsurrounding the cel l ,  thcy also have to enable uplake of substrates and extrusion of g'asteproducts. In other words only certain compounds are al lon'ed to pernteate through thatbarrier.Plasma rnembranes are composed of two rnain components: l ipids and proteins, the lattcrplay a very inlportant role in contact with the environment. Some rnembrane proteins areresponsible Íbrthe uptake of required compounds. There are two types of these proteins thatpart icipate in the transport of solutes and ions across the membrane. The l lrsr rype are poresand channels that làci l i tate the dif l i rsion of substrates without rcquir ing metabolic energy(Fig. lA). Thcy Íbrm structt lres that resernblc "holes" in the mcrnbrane. The second type arernembrane transportcrs: proteins that show a great varicty of transport rnechanisms,structures and specilicities. They are divided into 3 classes based on the energy required Íorthe transport process: ( i)  primary transporters (Fig. lB) use chemical cnergy released, i trrexample, in thc breakdown of ATP, ( i i )  secondary transpofters (Fig. lc) use cnergy storedin the electrochemical gradient of solutes and/or ions, ( i i i )  group translocators (Fig. lD)rnovc the solule and perfbrm its chernical rnodi l icat ion; the modiÍ led solute is released onthe clther side o1- the membrane. Mernbrane transpoftcrs interact with their substrates anclundergo structural changcs during the translocation process.The CitS protein is a sodiurn dcpendent citrate transporler lound in the pathogenic bacteriumKlebsiella pneunoniue. It belongs to the class ofsecondary transporters. It transports citratetogcther n' i th 2 sodium ions in the same direct ion ( into the bacterial cel l)  (Fig. lc).Therefore, it is termed a symporter, u,hich diÍïèrs liom the other types of secondarytransporters that eithcr movc substratcs in opposite directions (antiportcrs) or transpod onlyonc substrate independently of any coupling ion in one dircct ion (uniporter).  The transportolci trate is driven by the electrochernical gradient of the sodium ions across the nrembrane.This is knor.vn as the sodiurn ion motive Íbrce (smÍ).The CitS protein is very specif ic because i t  only recognises the citrate anion (citr-) as aslrbstratc. This suggests that thc structure ofthc protein has to be rvel l  aclapted to the role ofChapter  Vl lrcco-{nit ion, binding and translocation ol only this substrate. Studies of theÍirnction of thc transport proteins try to understand these processes takingrnolccular lcvcl and aim. ult imatcly, to cxplain the mechanisrn of transport.s1tï'ffi+lS , istructlrre andplace at theC-'hapter 4 of thydrophobictowards the c'Amino acid r,easi ly reachecthat the residuthe residues almemoFigure 2. l\(.Ía/a'r ) betwtornls a porrtrallslocatl0rVb corrcsprinver ted  or if  y  top lasmi rThc cytoplasnPart of that loprotein Íamil lStudies of th,shou ed that I(outside) sideÍiom thc perip(Chapter 3) reloop that ÍoldDcBA5S,Àb\^:I n  b - rATP )z,.r,rJ,.l-ï#r .,.,.:;:'-,r:ièr i di :--f pyruvate, - DIPEPl ' i gurc  l  D i Í Í c ren t  t rpes  o f t ranspor t  across  the  membrane:  (A)  channe l .  (B)  p r i r ra ry  t ranspor tc r .  (C)sccondarr transporter. (D) group translocator. Symbols and abbreviations: S substrate of thetransporter. A I 'P adcnosine 5'-triphosphate, ADP adenosine diphosphate. Pi Í iee phosphate group,P[P phosphocno lopyruvate ,  S-P phosphory , la tec l  sugar .  l lA .  l lB  and I IC  th ree  subun i ts  o Í '1he  sanrcpro te in ,  HPr  and EI  o thcr  cnz lmcs in lo l led  in  the  t rans lè r  o fphosphory l  g roup.Erery protein is bui l t  Í iom chains o1-amino acids of dif lerent lengths, cal led polypeptidechains. Such polypeptide chains Íbld into three-dinrensional structures, which give theproteins their specif ic functions. I lecause membrane proteins insert themselves into therrembrane rn a zigzag Íashion, part of the polypeptide chain fbnns the characterist ic hel icalstructure inside ol-the hydrophobic membrane environment.CitS consists of a 446 amino acids long polypeptide chain that traverses the membrane 11times and thus lbrms 1 I rnembrane-embedded parts (Fig. 2). These parts, namedlransmembrane segments (TMSs), Íbrm the hel ical structure. In contrast, the loops aremostly unstructr.rred and are local ised in the cytoplasm (inside) orperiplasm (outside) of the(Ciram-negative) bactcrial cel l .  In CitS there are a Íbu, large cytoplasmic loops, rvi th theIonsest onc connccting the TMS VI and VII (Fig. 2). The loop bctwccn TMS VIII  and IX(AH; Fig.2) is predicted by computational methods to also l i rrrn a hel ical structure. In134'ucture andlace at the)yruvate'ner. (C )r  of  the:e group.the samcrolypeptideh give thees into thestic hel ical:mbrane I IÍs, namedr loops areside) of thes, rvith the/ l l l  and  IX.ructure. InSummaryChapter 4 of this thcsis, i t  is shown that this loop is f i r lded as an arnphipathic hel ix, with thehydrophobic Íacc of the hel ix exposed tor 'vards the membranc and the hydrophil ic Íacetou'ards thc cytoplasm. This amphipathic Íèature was the key to the experirnental evidence.Amino acid residues at the hydrophobic Íàce of thc hel ix, close to the membrane, are noteasily reached by the u'ater-soluble reagcnts uscd to rnodily the residues. We havc shorvnthat the residues located on thc hydrophobic Íàce are not accessible Íbr such reagents, rvhi lethe residues at the hydrophil ic Íace arc.membraneFigure  2 .  Membrane topo log l  mode l  o f  the  C i ts  p ro te in .  TMSs are  ind ica ted  by  bores .  The loop AH(gre,t) betu'een TMS Vll l and IX tbrms an amphiprthic hclir. Loop Xa, the best-conserved region,lbrms a pore-loop stnlcturc and logclher s' ith TMS X and XI (t lu.shed bores\ is involved in thc substratetranslocation path['a1'. Trvo clsteine residues in the Xa region are represented as hlutk cirt lc.s. ScgmentVb corresponds kr a h1'drophobic rcgion: i l ' the protcin consists ol ' trvo homologous dorrains r,ithin \e r ted  o Í ien ta t ion .  each o i Í i ve  TMSs ( ind ica tcd  as  lushet l  àores) ,  Vb poss ib ly  fomrs  a  pore- loop.C l top lasnr ic  ( i i )  anc l  per ip lasmic  (oa l )  s ides  o f the  membranc  are  ind ica tcd .The cytoplasmic loop locatcd bctr.r,een TMS X and XI showed another unexpected structure.Part o1-that loop, cal led Xa, comprises the least changed region in the evolut ion of the CitSprotein Íàmily; i t  rvas postulatcd to be inr. 'olved in the binding of the substrate (Fig. 2).Studies of the trvo cysteine residues located in the cytoplasmic Xa region (Chapter 2)sho*'ed that they can be accessed by small  impermeable reagcnts from the periplasmic(outside) side of the membrane. Ho\\ 'evcr, largcrreagents were not able to access this regionÍiom the periplasm indicating that thcre is sorne size-restr icted cavity. More detai led studics(Chapter 3) revealed that this region Íonns a structure cal led a pore-loop, which is parl  of aloop that Íblds back into thc mcmbranc between the TMSs but never protrudes completcly1 3 5Chapter Vllto thc other side. This cxplains why undcr sortre condit ions. certain residues are easi lyaccessible Í iorn the other side of the n-rcmbrane. Pore-loop struclures are common inchannels but not in secondary transporters. Only in one làmily of secondary transporters, theglutanrate transp(n' lers, har. 'e pore-loops been clbsen ed in rcgions closely involr ed insuhstrate binding. l t  is thought that they lbnn a pathway Íbr the substrate and, by theirllexible structure, inducc and control thc changes in thc structure of the prcÍein aÍ1er thesubstrate is bound. As shou'n lbr ( ' i tS in Chapter I  of this thesis, binding of ci trate could, tosonre e\tent, inhibit  the accessibi l i t ) '  of the probcd residues. Surprisingly, complcteinhibit ion u'as achier"cd only in the prcscnce ot '  sodir-rm ions. Since sodiunr iorrs lre torrsmall  to causc pÍotection against the reagent by covering thc access to the rcsidues, astructural change in the protcin is probably thc main reason Íbr the observed effcct.One l irr ther interestin-u paft t l l - thc plotein is the large periplasrnic rcgion cal led Vb. The Vbsegrnent had bccn preriously prcdicted to lbrm a transmembranc segrnent lr l l t  cxpcrimentsshou'ed that i t  is exported into the periplasnr. A fecent analysis ol-the amino acid sequenceof thc secondary transpoftcrs'  prol i les in the CitS Íarni ly suggested the presence of trvodornir ins in thc protein structurc. For CitS, i t  is proposed (Chapter 1) that thc clonrainsconsist of Í lve TMSs and that TMS I is an addit ional scgnrent. According to the dornainnrrrdel,  region Xa is hornologous to rcgion Vb sLrggcsting that Vb could Íbrm another pore-lonp structure and that the two domajns have an inverted orientat ion in the rnembrane (Fig.a \Thc main object of thc research described in this thesis nas to obtain more inl irrrnationabor.rt  the structure ol ' thc CitS protein and i ts rclat ion to the function. The presence of theporc-loop in the structure is not accidental and therelbre lvc have tr ied to cxplain the role ofthis region in the translocation ol-the substrate. Besidcs, the role of thc arnphipathic hcl irAl l  and legions l ike Vb in the protein nrust be erplained. I t  is possible that such structuresare more corrnlon in transport protcins but rather diÍ ï lcult  to prcdict and local ise. Moreinlormation on membrane protein structurcs and transport mechanisms wil l  be helpful in thcÍuturc tbr a better understandin.e of the lunction and importance of these proteins Íor l iv ingorganrsrrs.Een essentiëlinhoud vanonderlroudenbuitenu e reldÍroeten z€ 0,Met anderedoorbreken.Plasmamenitspelen een btzijn verantrvtdezc eiwitterccrste tyllemetabole cnmembraan. lvertonen invr--rdeeld op I( i)  prirnairebi jvoorbeeldener_sle opgetranslocaticrtn'orden. Tratl  erandering rHct Cl i tS eiubacterie K1c.transportecrte i rv i t  to t  de 'dic hun subsonaÍhankelr j lVan cltraat wmernbraan. [--136

University of Groningen Research Database

  
 University of Groningen
Structural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniae
Sobczak - Elbourne, Iwona
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2005
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Sobczak - Elbourne, I. (2005). Structural and functional features of the Na+-citrate transporter CitS of
Klebsiella pneumoniae Groningen: s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
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Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
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Download date: 10-02-2018
VII
Summarv
One of thc essential structures rcquired by er"ery l iving organisnr is a rncmbranc that
encompasses the cytoplasm (the contents of the cel l) .  Membranes. not only have a very
important role in sustaining contacts with the outside rvorld, but also in protcct ing the cel l
against i t .  Whilst membranes have to be quite resistant to the stressfl l  enyironment
surrounding the cel l ,  thcy also have to enable uplake of substrates and extrusion of g'aste
products. In other words only certain compounds are al lon'ed to pernteate through that
barrier.
Plasma rnembranes are composed of two rnain components: l ipids and proteins, the lattcr
play a very inlportant role in contact with the environment. Some rnembrane proteins are
responsible Íbrthe uptake of required compounds. There are two types of these proteins that
part icipate in the transport of solutes and ions across the membrane. The l lrsr rype are pores
and channels that làci l i tate the dif l i rsion of substrates without rcquir ing metabolic energy
(Fig. lA). Thcy Íbrm structt lres that resernblc "holes" in the mcrnbrane. The second type are
rnembrane transportcrs: proteins that show a great varicty of transport rnechanisms,
structures and specilicities. They are divided into 3 classes based on the energy required Íor
the transport process: ( i)  primary transporters (Fig. lB) use chemical cnergy released, i trr
example, in thc breakdown of ATP, ( i i )  secondary transpofters (Fig. lc) use cnergy stored
in the electrochemical gradient of solutes and/or ions, ( i i i )  group translocators (Fig. lD)
rnovc the solule and perfbrm its chernical rnodi l icat ion; the modiÍ led solute is released on
the clther side o1- the membrane. Mernbrane transpoftcrs interact with their substrates ancl
undergo structural changcs during the translocation process.
The CitS protein is a sodiurn dcpendent citrate transporler lound in the pathogenic bacterium
Klebsiella pneunoniue. It belongs to the class ofsecondary transporters. It transports citrate
togcther n' i th 2 sodium ions in the same direct ion ( into the bacterial cel l)  (Fig. lc).
Therefore, it is termed a symporter, u,hich diÍïèrs liom the other types of secondary
transporters that eithcr movc substratcs in opposite directions (antiportcrs) or transpod only
onc substrate independently of any coupling ion in one dircct ion (uniporter).  The transport
olci trate is driven by the electrochernical gradient of the sodium ions across the nrembrane.
This is knor.vn as the sodiurn ion motive Íbrce (smÍ).
The CitS protein is very specif ic because i t  only recognises the citrate anion (citr-) as a
slrbstratc. This suggests that thc structure ofthc protein has to be rvel l  aclapted to the role of
Chapter  Vl l
rcco-{nit ion, binding and translocation ol only this substrate. Studies of the
Íirnction of thc transport proteins try to understand these processes taking
rnolccular lcvcl and aim. ult imatcly, to cxplain the mechanisrn of transport.
s1
tï'ffi
+l
S , i
structlrre and
place at the
C-'hapter 4 of t
hydrophobic
towards the c'
Amino acid r,
easi ly reachec
that the residu
the residues al
memo
Figure 2. l\
(.Ía/a'r ) betw
tornls a porr
trallslocatl0r
Vb corrcspr
inver ted  or i
f  y  top lasmi r
Thc cytoplasn
Part of that lo
protein Íamil l
Studies of th,
shou ed that I
(outside) side
Íiom thc perip
(Chapter 3) re
loop that Íold
DcBA
5
S
,Àb\^:
I n  b - r
ATP )z
,.r,rJ,.
l-ï#r .,.,.:;:'-,r:ièr i di :--f pyruvate
, - D
I
PEP
l ' i gurc  l  D i Í Í c ren t  t rpes  o f t ranspor t  across  the  membrane:  (A)  channe l .  (B)  p r i r ra ry  t ranspor tc r .  (C)
sccondarr transporter. (D) group translocator. Symbols and abbreviations: S substrate of the
transporter. A I 'P adcnosine 5'-triphosphate, ADP adenosine diphosphate. Pi Í iee phosphate group,
P[P phosphocno lopyruvate ,  S-P phosphory , la tec l  sugar .  l lA .  l lB  and I IC  th ree  subun i ts  o Í '1he  sanrc
pro te in ,  HPr  and EI  o thcr  cnz lmcs in lo l led  in  the  t rans lè r  o fphosphory l  g roup.
Erery protein is bui l t  Í iom chains o1-amino acids of dif lerent lengths, cal led polypeptide
chains. Such polypeptide chains Íbld into three-dinrensional structures, which give the
proteins their specif ic functions. I lecause membrane proteins insert themselves into the
rrembrane rn a zigzag Íashion, part of the polypeptide chain fbnns the characterist ic hel ical
structure inside ol-the hydrophobic membrane environment.
CitS consists of a 446 amino acids long polypeptide chain that traverses the membrane 11
times and thus lbrms 1 I rnembrane-embedded parts (Fig. 2). These parts, named
lransmembrane segments (TMSs), Íbrm the hel ical structure. In contrast, the loops are
mostly unstructr.rred and are local ised in the cytoplasm (inside) orperiplasm (outside) of the
(Ciram-negative) bactcrial cel l .  In CitS there are a Íbu, large cytoplasmic loops, rvi th the
Ionsest onc connccting the TMS VI and VII (Fig. 2). The loop bctwccn TMS VIII  and IX
(AH; Fig.2) is predicted by computational methods to also l i rrrn a hel ical structure. In
134
'ucture and
lace at the
)yruvate
'ner. (C )
r  of  the
:e group.
the samc
rolypeptide
h give the
es into the
stic hel ical
:mbrane I I
Ís, named
r loops are
side) of the
s, rvith the
/ l l l  and  IX
.ructure. In
Summary
Chapter 4 of this thcsis, i t  is shown that this loop is f i r lded as an arnphipathic hel ix, with the
hydrophobic Íacc of the hel ix exposed tor 'vards the membranc and the hydrophil ic Íace
tou'ards thc cytoplasm. This amphipathic Íèature was the key to the experirnental evidence.
Amino acid residues at the hydrophobic Íàce of thc hel ix, close to the membrane, are not
easily reached by the u'ater-soluble reagcnts uscd to rnodily the residues. We havc shorvn
that the residues located on thc hydrophobic Íàce are not accessible Íbr such reagents, rvhi le
the residues at the hydrophil ic Íace arc.
membrane
Figure  2 .  Membrane topo log l  mode l  o f  the  C i ts  p ro te in .  TMSs are  ind ica ted  by  bores .  The loop AH
(gre,t) betu'een TMS Vll l and IX tbrms an amphiprthic hclir. Loop Xa, the best-conserved region,
lbrms a pore-loop stnlcturc and logclher s' ith TMS X and XI (t lu.shed bores\ is involved in thc substrate
translocation path['a1'. Trvo clsteine residues in the Xa region are represented as hlutk cirt lc.s. Scgment
Vb corresponds kr a h1'drophobic rcgion: i l ' the protcin consists ol ' trvo homologous dorrains r,ith
in \e r ted  o Í ien ta t ion .  each o i Í i ve  TMSs ( ind ica tcd  as  lushet l  àores) ,  Vb poss ib ly  fomrs  a  pore- loop.
C l top lasnr ic  ( i i )  anc l  per ip lasmic  (oa l )  s ides  o f the  membranc  are  ind ica tcd .
The cytoplasmic loop locatcd bctr.r,een TMS X and XI showed another unexpected structure.
Part o1-that loop, cal led Xa, comprises the least changed region in the evolut ion of the CitS
protein Íàmily; i t  rvas postulatcd to be inr. 'olved in the binding of the substrate (Fig. 2).
Studies of the trvo cysteine residues located in the cytoplasmic Xa region (Chapter 2)
sho*'ed that they can be accessed by small  impermeable reagcnts from the periplasmic
(outside) side of the membrane. Ho\\ 'evcr, largcrreagents were not able to access this region
Íiom the periplasm indicating that thcre is sorne size-restr icted cavity. More detai led studics
(Chapter 3) revealed that this region Íonns a structure cal led a pore-loop, which is parl  of a
loop that Íblds back into thc mcmbranc between the TMSs but never protrudes completcly
1 3 5
Chapter Vll
to thc other side. This cxplains why undcr sortre condit ions. certain residues are easi ly
accessible Í iorn the other side of the n-rcmbrane. Pore-loop struclures are common in
channels but not in secondary transporters. Only in one làmily of secondary transporters, the
glutanrate transp(n' lers, har. 'e pore-loops been clbsen ed in rcgions closely involr ed in
suhstrate binding. l t  is thought that they lbnn a pathway Íbr the substrate and, by their
llexible structure, inducc and control thc changes in thc structure of the prcÍein aÍ1er the
substrate is bound. As shou'n lbr ( ' i tS in Chapter I  of this thesis, binding of ci trate could, to
sonre e\tent, inhibit  the accessibi l i t ) '  of the probcd residues. Surprisingly, complcte
inhibit ion u'as achier"cd only in the prcscnce ot '  sodir-rm ions. Since sodiunr iorrs lre torr
small  to causc pÍotection against the reagent by covering thc access to the rcsidues, a
structural change in the protcin is probably thc main reason Íbr the observed effcct.
One l irr ther interestin-u paft t l l - thc plotein is the large periplasrnic rcgion cal led Vb. The Vb
segrnent had bccn preriously prcdicted to lbrm a transmembranc segrnent lr l l t  cxpcriments
shou'ed that i t  is exported into the periplasnr. A fecent analysis ol-the amino acid sequence
of thc secondary transpoftcrs'  prol i les in the CitS Íarni ly suggested the presence of trvo
dornir ins in thc protein structurc. For CitS, i t  is proposed (Chapter 1) that thc clonrains
consist of Í lve TMSs and that TMS I is an addit ional scgnrent. According to the dornain
nrrrdel,  region Xa is hornologous to rcgion Vb sLrggcsting that Vb could Íbrm another pore-
lonp structure and that the two domajns have an inverted orientat ion in the rnembrane (Fig.
a \
Thc main object of thc research described in this thesis nas to obtain more inl irrrnation
abor.rt  the structure ol ' thc CitS protein and i ts rclat ion to the function. The presence of the
porc-loop in the structure is not accidental and therelbre lvc have tr ied to cxplain the role of
this region in the translocation ol-the substrate. Besidcs, the role of thc arnphipathic hcl ir
Al l  and legions l ike Vb in the protein nrust be erplained. I t  is possible that such structures
are more corrnlon in transport protcins but rather diÍ ï lcult  to prcdict and local ise. More
inlormation on membrane protein structurcs and transport mechanisms wil l  be helpful in thc
Íuturc tbr a better understandin.e of the lunction and importance of these proteins Íor l iv ing
organrsrrs.
Een essentiël
inhoud van
onderlrouden
buitenu e reld
Íroeten z€ 0,
Met andere
doorbreken.
Plasmamenit
spelen een bt
zijn verantrvt
dezc eiwitter
ccrste tylle
metabole cn
membraan. l
vertonen in
vr--rdeeld op I
( i)  prirnaire
bi jvoorbeeld
ener_sle opge
translocatic
rtn'orden. Trat
l  erandering r
Hct Cl i tS eiu
bacterie K1c.
transportecrt
e i rv i t  to t  de '
dic hun subs
onaÍhankelr j l
Van cltraat w
mernbraan. [--
136


University of Groningen

   University of GroningenStructural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniaeSobczak - Elbourne, IwonaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Sobczak - Elbourne, I. (2005). Structural and functional features of the Na+-citrate transporter CitS ofKlebsiella pneumoniae. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 26-12-2020VIISummarvOne of thc essential structures rcquired by er"ery l iving organisnr is a rncmbranc thatencompasses the cytoplasm (the contents of the cel l) .  Membranes. not only have a veryimportant role in sustaining contacts with the outside rvorld, but also in protcct ing the cel lagainst i t .  Whilst membranes have to be quite resistant to the stressfl l  enyironmentsurrounding the cel l ,  thcy also have to enable uplake of substrates and extrusion of g'asteproducts. In other words only certain compounds are al lon'ed to pernteate through thatbarrier.Plasma rnembranes are composed of two rnain components: l ipids and proteins, the lattcrplay a very inlportant role in contact with the environment. Some rnembrane proteins areresponsible Íbrthe uptake of required compounds. There are two types of these proteins thatpart icipate in the transport of solutes and ions across the membrane. The l lrsr rype are poresand channels that làci l i tate the dif l i rsion of substrates without rcquir ing metabolic energy(Fig. lA). Thcy Íbrm structt lres that resernblc "holes" in the mcrnbrane. The second type arernembrane transportcrs: proteins that show a great varicty of transport rnechanisms,structures and specilicities. They are divided into 3 classes based on the energy required Íorthe transport process: ( i)  primary transporters (Fig. lB) use chemical cnergy released, i trrexample, in thc breakdown of ATP, ( i i )  secondary transpofters (Fig. lc) use cnergy storedin the electrochemical gradient of solutes and/or ions, ( i i i )  group translocators (Fig. lD)rnovc the solule and perfbrm its chernical rnodi l icat ion; the modiÍ led solute is released onthe clther side o1- the membrane. Mernbrane transpoftcrs interact with their substrates anclundergo structural changcs during the translocation process.The CitS protein is a sodiurn dcpendent citrate transporler lound in the pathogenic bacteriumKlebsiella pneunoniue. It belongs to the class ofsecondary transporters. It transports citratetogcther n' i th 2 sodium ions in the same direct ion ( into the bacterial cel l)  (Fig. lc).Therefore, it is termed a symporter, u,hich diÍïèrs liom the other types of secondarytransporters that eithcr movc substratcs in opposite directions (antiportcrs) or transpod onlyonc substrate independently of any coupling ion in one dircct ion (uniporter).  The transportolci trate is driven by the electrochernical gradient of the sodium ions across the nrembrane.This is knor.vn as the sodiurn ion motive Íbrce (smÍ).The CitS protein is very specif ic because i t  only recognises the citrate anion (citr-) as aslrbstratc. This suggests that thc structure ofthc protein has to be rvel l  aclapted to the role ofChapter  Vl lrcco-{nit ion, binding and translocation ol only this substrate. Studies of theÍirnction of thc transport proteins try to understand these processes takingrnolccular lcvcl and aim. ult imatcly, to cxplain the mechanisrn of transport.s1tï'ffi+lS , istructlrre andplace at theC-'hapter 4 of thydrophobictowards the c'Amino acid r,easi ly reachecthat the residuthe residues almemoFigure 2. l\(.Ía/a'r ) betwtornls a porrtrallslocatl0rVb corrcsprinver ted  or if  y  top lasmi rThc cytoplasnPart of that loprotein Íamil lStudies of th,shou ed that I(outside) sideÍiom thc perip(Chapter 3) reloop that ÍoldDcBA5S,Àb\^:I n  b - rATP )z,.r,rJ,.l-ï#r .,.,.:;:'-,r:ièr i di :--f pyruvate, - DIPEPl ' i gurc  l  D i Í Í c ren t  t rpes  o f t ranspor t  across  the  membrane:  (A)  channe l .  (B)  p r i r ra ry  t ranspor tc r .  (C)sccondarr transporter. (D) group translocator. Symbols and abbreviations: S substrate of thetransporter. A I 'P adcnosine 5'-triphosphate, ADP adenosine diphosphate. Pi Í iee phosphate group,P[P phosphocno lopyruvate ,  S-P phosphory , la tec l  sugar .  l lA .  l lB  and I IC  th ree  subun i ts  o Í '1he  sanrcpro te in ,  HPr  and EI  o thcr  cnz lmcs in lo l led  in  the  t rans lè r  o fphosphory l  g roup.Erery protein is bui l t  Í iom chains o1-amino acids of dif lerent lengths, cal led polypeptidechains. Such polypeptide chains Íbld into three-dinrensional structures, which give theproteins their specif ic functions. I lecause membrane proteins insert themselves into therrembrane rn a zigzag Íashion, part of the polypeptide chain fbnns the characterist ic hel icalstructure inside ol-the hydrophobic membrane environment.CitS consists of a 446 amino acids long polypeptide chain that traverses the membrane 11times and thus lbrms 1 I rnembrane-embedded parts (Fig. 2). These parts, namedlransmembrane segments (TMSs), Íbrm the hel ical structure. In contrast, the loops aremostly unstructr.rred and are local ised in the cytoplasm (inside) orperiplasm (outside) of the(Ciram-negative) bactcrial cel l .  In CitS there are a Íbu, large cytoplasmic loops, rvi th theIonsest onc connccting the TMS VI and VII (Fig. 2). The loop bctwccn TMS VIII  and IX(AH; Fig.2) is predicted by computational methods to also l i rrrn a hel ical structure. In134'ucture andlace at the)yruvate'ner. (C )r  of  the:e group.the samcrolypeptideh give thees into thestic hel ical:mbrane I IÍs, namedr loops areside) of thes, rvith the/ l l l  and  IX.ructure. InSummaryChapter 4 of this thcsis, i t  is shown that this loop is f i r lded as an arnphipathic hel ix, with thehydrophobic Íacc of the hel ix exposed tor 'vards the membranc and the hydrophil ic Íacetou'ards thc cytoplasm. This amphipathic Íèature was the key to the experirnental evidence.Amino acid residues at the hydrophobic Íàce of thc hel ix, close to the membrane, are noteasily reached by the u'ater-soluble reagcnts uscd to rnodily the residues. We havc shorvnthat the residues located on thc hydrophobic Íàce are not accessible Íbr such reagents, rvhi lethe residues at the hydrophil ic Íace arc.membraneFigure  2 .  Membrane topo log l  mode l  o f  the  C i ts  p ro te in .  TMSs are  ind ica ted  by  bores .  The loop AH(gre,t) betu'een TMS Vll l and IX tbrms an amphiprthic hclir. Loop Xa, the best-conserved region,lbrms a pore-loop stnlcturc and logclher s' ith TMS X and XI (t lu.shed bores\ is involved in thc substratetranslocation path['a1'. Trvo clsteine residues in the Xa region are represented as hlutk cirt lc.s. ScgmentVb corresponds kr a h1'drophobic rcgion: i l ' the protcin consists ol ' trvo homologous dorrains r,ithin \e r ted  o Í ien ta t ion .  each o i Í i ve  TMSs ( ind ica tcd  as  lushet l  àores) ,  Vb poss ib ly  fomrs  a  pore- loop.C l top lasnr ic  ( i i )  anc l  per ip lasmic  (oa l )  s ides  o f the  membranc  are  ind ica tcd .The cytoplasmic loop locatcd bctr.r,een TMS X and XI showed another unexpected structure.Part o1-that loop, cal led Xa, comprises the least changed region in the evolut ion of the CitSprotein Íàmily; i t  rvas postulatcd to be inr. 'olved in the binding of the substrate (Fig. 2).Studies of the trvo cysteine residues located in the cytoplasmic Xa region (Chapter 2)sho*'ed that they can be accessed by small  impermeable reagcnts from the periplasmic(outside) side of the membrane. Ho\\ 'evcr, largcrreagents were not able to access this regionÍiom the periplasm indicating that thcre is sorne size-restr icted cavity. More detai led studics(Chapter 3) revealed that this region Íonns a structure cal led a pore-loop, which is parl  of aloop that Íblds back into thc mcmbranc between the TMSs but never protrudes completcly1 3 5Chapter Vllto thc other side. This cxplains why undcr sortre condit ions. certain residues are easi lyaccessible Í iorn the other side of the n-rcmbrane. Pore-loop struclures are common inchannels but not in secondary transporters. Only in one làmily of secondary transporters, theglutanrate transp(n' lers, har. 'e pore-loops been clbsen ed in rcgions closely involr ed insuhstrate binding. l t  is thought that they lbnn a pathway Íbr the substrate and, by theirllexible structure, inducc and control thc changes in thc structure of the prcÍein aÍ1er thesubstrate is bound. As shou'n lbr ( ' i tS in Chapter I  of this thesis, binding of ci trate could, tosonre e\tent, inhibit  the accessibi l i t ) '  of the probcd residues. Surprisingly, complcteinhibit ion u'as achier"cd only in the prcscnce ot '  sodir-rm ions. Since sodiunr iorrs lre torrsmall  to causc pÍotection against the reagent by covering thc access to the rcsidues, astructural change in the protcin is probably thc main reason Íbr the observed effcct.One l irr ther interestin-u paft t l l - thc plotein is the large periplasrnic rcgion cal led Vb. The Vbsegrnent had bccn preriously prcdicted to lbrm a transmembranc segrnent lr l l t  cxpcrimentsshou'ed that i t  is exported into the periplasnr. A fecent analysis ol-the amino acid sequenceof thc secondary transpoftcrs'  prol i les in the CitS Íarni ly suggested the presence of trvodornir ins in thc protein structurc. For CitS, i t  is proposed (Chapter 1) that thc clonrainsconsist of Í lve TMSs and that TMS I is an addit ional scgnrent. According to the dornainnrrrdel,  region Xa is hornologous to rcgion Vb sLrggcsting that Vb could Íbrm another pore-lonp structure and that the two domajns have an inverted orientat ion in the rnembrane (Fig.a \Thc main object of thc research described in this thesis nas to obtain more inl irrrnationabor.rt  the structure ol ' thc CitS protein and i ts rclat ion to the function. The presence of theporc-loop in the structure is not accidental and therelbre lvc have tr ied to cxplain the role ofthis region in the translocation ol-the substrate. Besidcs, the role of thc arnphipathic hcl irAl l  and legions l ike Vb in the protein nrust be erplained. I t  is possible that such structuresare more corrnlon in transport protcins but rather diÍ ï lcult  to prcdict and local ise. Moreinlormation on membrane protein structurcs and transport mechanisms wil l  be helpful in thcÍuturc tbr a better understandin.e of the lunction and importance of these proteins Íor l iv ingorganrsrrs.Een essentiëlinhoud vanonderlroudenbuitenu e reldÍroeten z€ 0,Met anderedoorbreken.Plasmamenitspelen een btzijn verantrvtdezc eiwitterccrste tyllemetabole cnmembraan. lvertonen invr--rdeeld op I( i)  prirnairebi jvoorbeeldener_sle opgetranslocaticrtn'orden. Tratl  erandering rHct Cl i tS eiubacterie K1c.transportecrte i rv i t  to t  de 'dic hun subsonaÍhankelr j lVan cltraat wmernbraan. [--136

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Structural and functional features of the Na+-citrate transporter CitS of Klebsiella pneumoniae

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