Structural characterization of core-bradavidin in complex with biotin

RESEARCH ARTICLE S uc u al cha ac e iza ion o co e-b ada idin in complex wi h bio in Ni in Ag awal 1 , Juha A. E. Ma ¨a ¨ a ¨ 2,3 , Ma kku S. Kulomaa 2 , Vesa P. Hy o ¨nen 2,3 , Ma k S. Johnson 1 , Tomi T. Ai enne 1 * 1S uc u al Bioin o ma ics Labo a o y, Biochemis y, Facul y o Science and Enginee ing, Åbo Akademi Uni e si y, Tu ku, Finland, 2Facul y o Medicine and Li e Sciences and BioMediTech, Uni e si y o Tampe e, Tampe e, Finland, 3Fimlab Labo a o ies, Tampe e, Finland * omi.ai enne@abo. i Abs ac B ada idin is a e ame ic bio in-binding p o ein simila o chicken a idin and bac e ial s ep- a idin, and was o iginally cloned om he ni ogen- ixing bac e ia B ady hizobium diazoe i- ciens. We ha e p e iously epo ed he c ys al s uc u e o he ull-leng h, wild- ype (w ) b ada idin wi h 138 amino acids, whe e he C- e minal esidues Gly129-Lys138 (“B ad- ag”) ac as an in insic ligand (i.e. Gly129-Lys138 bind in o he bio in-binding si e o an adjacen subuni wi hin he same e ame ) and has po en ial as an a ini y ag o bio echnological pu poses. He e, he X- ay s uc u e o co e-b ada idin lacking he C- e minal esidues Gly114-Lys138, and hence missing he B ad- ag, was c ys allized in complex wi h bio in a 1.60 Å esolu ion [PDB:4BBO]. We also epo a homology model o hoda idin, an a idin- like p o ein om Rhodopseudomonas palus is, and o an a idin-like p o ein om B ady hi- zobium sp. Ai1a-2, bo h o which ha e he B ad- ag sequence a hei C- e minus. Mo eo e , co e-b ada idin V1, an enginee ed a ian o he o iginal co e-b ada idin, was also exp essed a high le els in E.coli, as well as a double mu an (Cys39Ala and Cys69Ala) o co e-b ada idin (CC mu an ). Ou da a help us o u he enginee he co e-b ada idin– B ad- ag pai o bio echnological assays and chemical biology applica ions, and p o ide deepe insigh in o he bio in-binding mode o b ada idin. In oduc ion A idins (A ds) a e p o eins p oduced in o iduc s o bi ds, ep iles and amphibians, and in se - e al di e en bac e ia [1,2]. In na u e, A ds a e mos s able in hei e ame ic [1,3–5] and dime ic [6] o ms. They ha e a high a ini y o D-bio in (K d = ~10 −15 M o chicken A d) [1,4,7], which makes hem a ac i e p o eins o nume ous bio echnological applica ions [7– 10]. The bes s udied A ds o da e a e he euka yo ic chicken A d [1–3,11] and he bac e ial s ep a idin om S ep omyces a idinii [5,12,13]. As a sec e ed p o ein, chicken A d is pos - ansla ionally modi ied by clea age o he 24 amino acid N- e minal signal pep ide and by gly- cosyla ion a Asn17; he esul ing ma u e p o ein has 128 amino acids [1,14]. The 159 amino acid gene p oduc o he ull-leng h s ep a idin, in u n, is na u ally immed a bo h he N PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Ci a ion: Ag awal N, Ma¨a¨ a¨JAE, Kulomaa MS, Hy o¨nen VP, Johnson MS, Ai enne TT (2017) S uc u al cha ac e iza ion o co e-b ada idin in complex wi h bio in. PLoS ONE 12(4): e0176086. h ps://doi.o g/10.1371/jou nal.pone.0176086 Edi o : Eugene A. Pe myako , Russian Academy o Medical Sciences, RUSSIAN FEDERATION Recei ed: Feb ua y 9, 2017 Accep ed: Ap il 5, 2017 Published: Ap il 20, 2017 Copy igh : ©2017 Ag awal e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. The coo dina es and s uc u e ac o s o he X- ay s uc u e o co e-b ada idin ha e been deposi ed in he P o ein Da a Bank wi h en y code 4BBO. Funding: This wo k was suppo ed by g an unding om he Academy o Finland (257814 (MSJ), 272283 (MSJ), 290506 (VPH), 261285 (MSK), and 272288 (MSK)); Sig id Juselius Founda ion; Joe Pen i, and To Bo g Memo ial Fund; Academy o Finland FIRI p og am (141398) and C e mini: he mos s udied o m has 127 amino acids, only con aining esidues 13–139 o he ull-leng h, and is e e ed o as co e-s ep a idin [5]. This na u ally occu ing unca ed o m o s ep a idin has been shown o ha e low agg ega e o ma ion and high solubili y, while e aining high a ini y o bio in [15]. Mo eo e , he c ys al s uc u e o ull-leng h s ep- a idin e ealed ha he 20- esidue C- e minal ex ension ( esidues 139–159) binds on he su - ace o he p o ein and ha esidues 150–153 occupy he ligand-binding si e o he same subuni —ac ing as an in asubuni in insic ligand [16]. In addi ion o chicken A d and s ep a idin, o he A ds ha e been cha ac e ized. They include he na u al euka yo ic A ds om, o example, zeb a ish (Danio a io) [17], og (Xenopus opicalis) [18], and mush oom (Pleu o us co nucopiae) [19], and he bac e ial A ds, such as he dime ic hiza idin (Rhizobium e li) [6], shwana idin (Shewanella deni i icans) [20] and hoe a idin (Hoe lea pho o ophica) [21]; he he mos able e ame ic bu ka idin (Bu - kholde ia pseudomallei) [22]; as well as b ada idin II (B ady hizobium diazoe iciens), which has a highly dynamic oligome ic s uc u e [23]. Apa om he na u ally occu ing A ds, a numbe o gene ically enginee ed A ds [7] ha e been p oduced, oo. These include he dual- chain A d (dcA d) [24] and single-chain A d (scA d) [25], espec i ely wi h wo and ou simul aneously modi iable ligand-binding si es, he monome ic s ep a idin [26–28], he s e- oid-binding A d (sbA d) [29,30] and an ex emely he mos able and p o ease esis an chi- me ic A d [31]. B ada idin is a e ame ic A d om a ni ogen- ixing bac e ium (B.diazoe iciens) ound in oo nodules o soy beans [32]. Wild- ype (w ) b ada idin, a e clea age o he 25- esidue sig- nal pep ide, has 138 amino acid esidues, o which he las en C- e minal esidues a e known as he B ad- ag [33]. The B ad- ag ( 129 GSEKLSNTKK) binds o he ligand-binding si e o a neighbo ing subuni and hence se es as an in insic, in e subuni ligand, dissimila bo h in mode o in e ac ion and sequence o he C- e minal sequence ound in ull-leng h s ep a idin [16] ha ac s as an in insic in asubuni ligand. The key esidues o B ad- ag in e ac ing wi h he ligand-binding si e a e Glu131, Lys132 and Leu133, whe eas in ull-leng h s ep a idin he key esidues a e Asn150, Gly151, Asn152, and P o153. In he case o s ep a idin, se e al pep- ide ags ha e been de eloped, including s ep- ag I [34], s ep- ag II [34,35], Nano- ag [36] and SBP- ag [37]; all o which ha e a di e en binding mode in compa ison o he B ad- ag. He e, we epo he e ame ic X- ay s uc u e o co e-b ada idin in complex wi h bio in a 1.60 Å esolu ion [PDB:4BBO]. In compa ison o he X- ay s uc u e o w b ada idin ( e a- me ; 138 amino acids/14 kDa pe subuni ), co e-b ada idin is a i icially unca ed a he C- e minus con aining only esidues 1–118 (12 kDa pe subuni ) and hence missing he B ad- ag [32]. In addi ion o hese b ada idin s uc u es, h ee s uc u es o b ada idin II (each 115 amino acids/13 kDa pe subuni ) a e known: wo di e en c ys alline o ms o he apo p o ein, he monome ic Fo m-A [PDB:4GGR] and he dime ic Fo m-B [PDB:4GGT], as well as a e a- me ic s uc u e in complex wi h bio in [PDB:4GGZ] [23]. All he known X- ay s uc u es o b ada idin and b ada idin II a e om he same bac e ium (B.diazoe iciens sp. no ; his s ain was ea lie known as he s ain USDA 110 o B ady hizobium japonicum [38]). The co e-b a- da idin s uc u e no only gi es insigh in o he de ailed bio in-binding mode o b ada idin bu also helps us o u he enginee co e-b ada idin as a ecep o wi h igh e binding owa ds ligands such as he B ad- ag i sel . We also epo homology models o he a idin-like p o eins om Rhodopseudomonas palus is ( hoda idin [39]) and B ady hizobium sp. Ai1a-2 ( e e ed o he e as b ada idin A2); bo h o hese p o eins ha e he B ad- ag sequence a hei C- e mi- nus. Ou be e unde s anding abou b ada idin binding o di e en ligands may aid in he de elopmen o no el cons uc s p o iding addi ional, imp o ed ools o bio echnological pu poses. Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 2 / 21 and Åbo Akademi Cen e o Excellence in Cell S ess and Aging. The unde s had no in luence in he s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Fimlab Labo a o ies (Tampe e, Finland) p o ided suppo in he o m o sala ies o au ho VPH and in he o m o esea ch ma e ials o he g oup lead by VPH, bu did no ha e any addi ional ole in he s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . The speci ic oles o hese au ho s a e a icula ed in he ‘au ho con ibu ions’ sec ion. Compe ing in e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . VPH is a ilia ed o Fimlab Labo a o ies; Fimlab Labo a o ies has suppo ed inancially he esea ch conduc ed in he esea ch g oup led by VPH. This does no al e ou adhe ence o PLoS ONE policies on sha ing da a and ma e ials. Resul s O e all s uc u e o co e-b ada idin The 3D s uc u e o co e-b ada idin in complex wi h bio in was sol ed a 1.60 Å esolu ion. This s uc u e ep esen s an a i icially unca ed o m o b ada idin and lacks he C- e minal esidues Gly114-Lys138 ( esidues Gly129-Lys138 co espond o B ad- ag) [32]. The sequence o he sol ed s uc u e sha es 35% iden i y wi h chicken A d [PDB:1AVD] [3], and he o e all e ame ic s uc u e and he olds o he indi idual subuni s I-IV (numbe ing acco ding o [4]) o co e-b ada idin a e ypical o A ds, including he w b ada idin s uc u e epo ed in [33] (Fig 1). We ha e also ied o c ys allize w b ada idin in complex wi h bio in wi hou suc- cess, which may ha e been due o he p esence o he bio in-compe ing C- e minal B ad- ag sequence in he w b ada idin. I is also possible ha econ igu a ion o he C- e minus o w b ada idin occu ed due o bio in binding and ha his may ha e al e ed c ys al con ac s and a ec ed c ys al o ma ion. Bio in-binding mode o co e-b ada idin—Conse ed ea u es Despi e he low sequence iden i y be ween co e-b ada idin and chicken A d, he deeply bu - ied esidues in ol ed in bio in binding and he mode o binding a e highly conse ed (Fig 2). Like in chicken A d [PDB:1AVD], he co e-b ada idin–bio in in e ac ion is s abilized by se - e al hyd ogen bonds (H-bonds) (Fig 2). In mo e de ail, 1) Asn9 Nδ(Asn12 in A d), Se 13 Oγ (Se 16) and Ty 31 Oη(Ty 33) all o m H-bonds wi h he 2´ oxygen a om o he u eido ing o bio in; 2) Asp107 Oδ(Asn118) o ms a H-bond in e ac ion wi h he 1´ ni ogen a om o he u eido ing; 3) Asn33 Oδ(Th 35) o ms a H-bond o he 3´ N a om o he u eido ing; 4) Th 77 Oγ(Th 77) has pola in e ac ions wi h he sul u a om o he e ahyd o hiophene ing; and 5) Se 75 Oγ(Se 75) o ms a H-bond o one oxygen a om o he ca boxyla e g oup o he ale ic acid moie y (b ada idin numbe ing acco ding o [40]). Th ee s uc u al wa e mole- cules (HOH73, HOH145 and HOH189) a e also loca ed close o he ca boxyla e end o bio in. Mo eo e , se e al conse ed hyd ophobic in e ac ions ypical o A ds a e also seen in co e- b ada idin and include he in e ac ion o bio in wi h T p89 (T p97 in A d) and T p99 (T p110; om ano he subuni ); hese esidues a e espec i ely 3.7 Åand 4.3 Ådis an om bio in. Bio in-binding mode o co e-b ada idin—Unique ea u es Asp107 and Asn33 o co e-b ada idin a e equi alen o Asn118 and Th 35 in A d [PDB: 1AVD]. The side-chain oxygen a om o each o hese esidues in e ac s wi h he 1´ and 2´ N a oms o bio in bu he o e all H-bonding ne wo k o he side chains o hese esidues wi h he su ounding esidues a ies be ween co e-b ada idin and A d (Fig 3). Asp107 in co e-b ada- idin is wi hin H-bonding dis ance o a s uc u al wa e molecule (HOH2014), Asn9, Gln10, T p75 (equi alen o Phe79 in A d), T p89 and Ala106, whe eas in A d only Asn12, Asp13, T p97 and Ile117 a e su icien ly close o he co esponding esidue Asn118. Bo h se s o esi- dues make hyd ophobic in e ac ions wi h he u eido ing moie y o bio in in co e-b ada idin and in A d. In b ada idin Ty 11 may be o special impo ance o ligand binding, since in he s uc u e o he bio in complex o co e-b ada idin i has mo ed signi ican ly in compa ison o he loca- ion in he w b ada idin s uc u e (see below). This esidue is also poo ly conse ed and, o ou knowledge, a y osine esidue a he equi alen posi ion is only ound in a ew bac e ial A ds, which includes he only o he epo ed B ad- ag con aining A d, hoda idin om Rho- dopseudomonas palus is [39], and he no el A d-like sequences ha we ha e iden i ied in he Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 3 / 21 gene a B ady hozibium,Meso hizobium,Bu kholde ia (Pseudomonas),Ca enulispo a and Ac i- noca enispo a (da a no shown). The Nδa om o Asn33 in co e-b ada idin is H-bonded o Se 38 Oγand Cys39 O (Fig 4), whe eas in chicken A d he equi alen H-bonds a e missing. Ou o he known c ys al s uc- u es o o he A ds, simila H-bond in e ac ions a e only seen in hiza idin [PDB:3EW2] and hoe a idin [PDB:4Z28]. In bo h s uc u es, an aspa agine esidue equi alen o Asn33 o co e- b ada idin is s abilized by H-bonds o he side-chain oxygen a om o a h eonine esidue (Se 38 Oγin co e-b ada idin) and o he main-chain oxygen a om o a glycine esidue (Cys39 Fig 1. Supe imposi ion o he Cα aces o subuni I o co e-b ada idin (o ange) [PDB:4BBO], w b ada idin (magen a) [PDB:2Y32] and chicken A d (cyan) [PDB:1AVD]. Fo cla i y, only he bio in bound o co e-b ada idin is shown (s ick model). The loop egions L1,2 o L7,8, and he N and C e mini, a e labelled. h ps://doi.o g/10.1371/jou nal.pone.0176086.g001 Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 4 / 21 O in co e-b ada idin). In w b ada idin, Asn33 is no connec ed o hese esidues bu ins ead H-bonds o Asn33 Nδand Asp40 Oδ, he o ien a ion o he Asp40 side chain being lipped in he opposi e di ec ion in compa ison o co e-b ada idin due o a ea angemen o he L3,4 loop. Mo eo e , in co e-b ada idin, he side chain o Asp40 is H-bonded o Leu67 N and Fig 2. Compa ison (s e eo iew) o he bio in-binding esidues (s icks) o co e-b ada idin (o ange; bold labels) [PDB:4BBO] and chicken A d (cyan; labels in b acke s) [PDB:1AVD]. The Cα aces we e supe imposed. T p99 (T p110 in A d) is shown om subuni III; o he esidues a e om subuni I. The bound bio in ligands a e d awn as hick s icks. Ni ogen a oms a e shown in blue, oxygen a oms in ed and sul u a oms in yellow. Wa e molecules a e d awn as ed sphe es and H-bonds o co e-b ada idin as g ey dashed lines (dis ances in Ångs o ¨ms; *= 2.8 Å). h ps://doi.o g/10.1371/jou nal.pone.0176086.g002 Fig 3. In e ac ions o Asp107 o co e-b ada idin (o ange; bold labels) and he equi alen Asn118 o chicken A d (cyan; labels in b acke s). Se en H-bonds s abilize he side chain o Asp107 in co e-b ada idin (a), whe eas only i e H-bonds s abilize he equi alen Asn118 in chicken A d (b). Non-ca bon a om colou ing as in Fig 2. H-bonds a e d awn as g ey dashed lines o co e-b ada idin (a) and blue dashed lines o chicken A d (b); dis ances in Ångs o ¨ms. h ps://doi.o g/10.1371/jou nal.pone.0176086.g003 Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 5 / 21 Gly68 N o he L5,6 loop, o Lys43 Nzo he L3,4 loop, and o Cys69 S (L5,6 loop) ha o ms he disul ide b idge wi h Cys39 (Fig 5). The side-chain o ien a ion o Glu41 is also lipped o he opposi e di ec ion in he co e-b ada idin s uc u e (closed L3,4 loop) e sus he w b ada- idin s uc u e (open L3,4 loop), and s abilized by di e en in e ac ions (Fig 6). Hence, Asp40 Fig 4. In e ac ions o Asn33 o co e-b ada idin (o ange; bold labels) and he equi alen Th 35 o chicken A d (cyan; labels in b acke s). Asn33 o co e-b ada idin is H-bonded (g ey dashed lines) o Se 38 and Cys39, whe eas in chicken A d he equi alen H-bonds canno be o med. Bio in molecules o bo h p o eins a e shown as hin s icks. Non- ca bon a oms a e colou ed as in Fig 2. Dis ances a e shown in Ångs o ¨ms. h ps://doi.o g/10.1371/jou nal.pone.0176086.g004 Fig 5. Compa ison o Asp40 in co e- (o ange; bold labels) [PDB:4BBO] and w b ada idin (magen a; labels in b acke s) [PDB:2Y32]. In co e- b ada idin (a), he side chain o Asp40 is lipped o an opposi e di ec ion as compa ed o w b ada idin (b). Bio in (a) and esidues K132 and L133 (b) occupying he same space as bio in in co e-b ada idin (see a) a e shown as sphe es. Non-ca bon a oms a e colou ed as in Fig 2. H-bonds a e shown as dashed lines; dis ances in Ångs o ¨ms. The weigh ed 2Fo-Fc elec on densi y map a ound Asp40 (a, b) is shown as a blue mesh (con ou le el o 1.0 σ). h ps://doi.o g/10.1371/jou nal.pone.0176086.g005 Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 6 / 21 and Glu41 may ha e a i al ole o ligand binding and in s abilizing he unique L3,4 loop con- o ma ions o w b ada idin (B ad- ag as ligand) and co e-b ada idin (bio in as ligand), wo a ia ions o he same p o ein wi h qui e di e en bound ligands. In compa ison wi h T p99 in w b ada idin, in co e-b ada idin he yp ophan esidue mo es by 2 Å, enabling he esidue o be e seal he ligand-binding pocke con aining bio in (see below). An Ångs o¨m-scale shi in a omic posi ion is also seen o Se 38 o co e-b ada i- din [PDB:4BBO] in compa ison o w b ada idin [PDB:2Y32]. The mo emen o Se 38 mim- ics he “pinching e ec ” epo ed o he equi alen h eonine esidue in hoe a idin [21] [PDB:4Z6J, 4Z28] and hiza idin [41] [PDB: 3EW1, 3EW2]; bo h Th 55 o hoe a idin and Th 48 o hiza idin o he L3,4-loop espec i ely mo e closely app oach Leu113 and Leu104 (loca ed on he β7-s and; Leu91 in b ada idin) as a esul o bio in binding (a simila “pinch- ing e ec ” can also be seen in shwana idin [20] [PDB:3SZH, 3SZJ] and b ada idin II [23] [PDB:4GGT, 4GGZ]). In each o hese s uc u es, he leucine side chains occupy he same ela- i e loca ions, sugges ing ha he pinching e ec migh no be es ic ed only o dime ic A ds and, in gene al, migh e lec an adap a ion o he L3,4-loop o ligand binding. The a oma ic esidues T p70 and Phe79 o chicken A d a e espec i ely eplaced by Phe66 and T p75 in b ada idin. E en sub le di e ences such as hese may ha e an e ec on he lexi- bili y o he L5,6 loop and he bio in-binding p ope ies o co e-b ada idin: in A d, T p70 Nη is H-bonded o Th 77 Oγ; whe eas, in b ada idin, an equi alen H-bonding in e ac ion is missing. To ou knowledge, all o he bac e ial A ds iden i ied so a —including bo h dime ic and e ame ic p o eins—ha e a yp ophan esidue a he posi ion equi alen o T p75 o b a- da idin, whe eas all o he cha ac e ized euka yo ic A ds ha e a phenylalanine esidue a his posi ion. The disul ide b idge o b ada idin—Unusual con igu a ion The L3,4 loop o e ame ic co e-b ada idin and o w b ada idin is s abilized by a disul ide b idge be ween esidues Cys39 (L3,4 loop) and Cys69 (L5,6 loop), simila ly o dime ic hiza i- din [PDB:3EW2] [41], shwana idin [PDB:3SZJ] [20] and hoe a idin [PDB:4Z28] [21], as well as b ada idin II [PDB:4GGZ] wi h i s highly dynamic oligome ic s uc u e [23]. In b ada idin, Fig 6. Compa ison (s e eo iew) o Glu41 in co e-b ada idin (o ange; bold labels) [PDB:4BBO] and w b ada idin (magen a; labels in b acke s) [PDB:2Y32]. In co e-b ada idin, he side chain o Glu41 is H- bonded (g ey dashed lines) o se e al neighbo ing esidues and is o ien ed in he opposi e di ec ion as compa ed o w b ada idin, whe e he side chain o Glu41 is acing he sol en and H-bonded (blue dashed line) only o Lys137. A pa o he acyl moie y o he bound bio in (BTN) o he co e-b ada idin s uc u e is shown as ligh g ey s icks. Non-ca bon a om colou ing as in Fig 2. Wa e molecules a e d awn as small ed sphe es. Dis ances a e shown in Ångs o ¨ms. h ps://doi.o g/10.1371/jou nal.pone.0176086.g006 Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 7 / 21 howe e , cys eine Cys39 o he L3,4 loop is loca ed one esidue ea lie in he sequence, as epo ed ecen ly by A aham e al. (2015) o hoe a idin [21]. Thus, Cys39 is no exac ly s uc u ally equi alen o Cys50 in hiza idin, Cys45 in shwana idin, Cys57 in hoe a idin and Cyss44 in b ada idin II, whe eas he o he cys eine o he disul ide bond wi hin he L5,6 loop, Cys69 in b ada idin, is conse ed despi e he ac ha he con o ma ion o he L5,6 loop a ies wi hin hese p o eins. Mo eo e , in he case o he bio in-complex s uc u e o co e-b ada i- din, he ea lie posi ion o he cys eine esidue does no seem o clea ly a ec he con o ma ion o he L3,4 loop, which is in a simila con o ma ion in all o he bio in complex s uc u es lis ed abo e. In compa ison o he w b ada idin s uc u e, which lacks bio in bu has he B ad- ag sequence wi hin he ligand-binding pocke , he con igu a ion o he disul ide b idge o co e- b ada idin is, howe e , al e ed and di ec ly ela ed o he posi ion and con o ma ion o Cys39 (Fig 7). As desc ibed abo e, he esidue adjacen o Cys39 in b ada idin, Asp40, may ha e a special impo ance he e, oo, because he side chain is lipped in he opposi e di ec ion in co e-b ada idin e sus w b ada idin. I is no ye known how he B ad- ag i sel a ec s he con o ma ion o he L3,4 loop when and i bio in we e bound in he in ac w s uc u e, since we ha e no been no able o c ys allize w b ada idin in complex wi h bio in. All in all, he con igu a ion o he disul ide bond in bo h o ms o b ada idin, co e- wi h bio in and w sans bio in, a e unique, and likely do ep esen he unique s uc u al ea u es needed o enable he p esence and ecogni ion o wo di e en ligands by he b ada idin s uc u e. Fig 7. Supe imposi ion o he esidues 35–41 (L3,4 loop) o co e-b ada idin (o ange; bold labels) [PDB:4BBO] and w b ada idin (magen a; labels in b acke s) [PDB:2Y32]. h ps://doi.o g/10.1371/jou nal.pone.0176086.g007 Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 8 / 21 Subuni in e aces A ds a e s abile o e a wide ange o condi ions, including empe a u e and pH. The co e-b a- da idin–bio in complex wi h T m = 97.9±0.2˚C is less s able han chicken A d (T m wi h bound bio in 118˚C), s ep a idin (T m wi h bound bio in 112˚C), [32] and e en w b ada idin (T m wi hou bio in = 96.2±0.1˚C, and 101.7±0.1˚C wi h bio in) [33]. The key in e ac ions esponsible o s abili y a e ound a he subuni in e aces, which can be di ided in o h ee majo ca ego ies: he in e ace be ween subuni s I and II (IF1,2), be ween subuni s I and III (IF1,3) and be ween subuni s I and IV (IF1,4 in e ace); in he dime ic A ds, only he IF1,4 in e ace is p esen . All o he subuni -subuni in e aces o co e-b ada idin and w b ada idin a e s uc u ally highly simila . A key in e acial esidue—Ty 90 om each o he ou subuni s—is loca ed a he cen e o he e ame in bo h o ms o b ada idin, whe eas in chicken A d [PDB:1AVD] and s ep a idin [PDB: 3YR2] he esidues equi alen o Ty 90 a e espec i ely Leu98 and Leu109. Since we ha e ecen ly published he de ailed analysis o he subuni in e ace o w b ada idin [33], we will ocus he e only on egions unique o co e-b ada idin as compa ed o w b ada idin. A he IF1,2 in e ace o co e-b ada idin, he posi ion o T p99 (subuni II; equi alen o T p110 o chicken A d) a he ip o he L7,8 loop and he spa ial a angemen o T p99 wi h espec o Ty 11 (subuni I) di e by o e an Ångs o¨m in compa ison o w b ada- idin (S1 Fig). These di e ences, oge he wi h he con o ma ional adap a ion o he L3,4 loop, a e he majo di e ences ha help hese b ada idin s uc u es ecognize wo e y di e en ligands, bio in and he B ad- ag. The C- e minal B ad- ag sequence en e s he ligand-binding pocke o w b ada idin be ween Ty 11 and T p99, and Ty 11 Oη(subuni I) o ms a H-bond- ing in e ac ion wi h Se 130 N (3.4 Å; subuni III) o he B ad- ag sequence and wi h one s uc- u al wa e molecule (HOH2027). In co e-b ada idin, Ty 11 OηH-bonds o h ee wa e molecules (HOH2014, HOH2015 and HOH2017). The co e- o he IF1,3 in e ace in he co e- and w b ada idin s uc u es is o med by esi- dues Gln86, Leu88, Ty 90, Ala104 and Ala106, and is s uc u ally highly simila . The IF1,4 in e ace is also e y simila in bo h s uc u es: i is clea ly he la ges in e ace in e ms o con- ac a ea and he numbe o esidues in ol ed—47 in w b ada idin [33]—and bo h he co e- and w b ada idin s uc u es a e s abilized by a ious non-co alen in e ac ions as lis ed in [33]. The L7,8 loops, howe e , ha e di e en con o ma ions since he esidues Gly57-Ty 63 a e in con ac wi h and adap o he binding o bio in o co e-b ada idin and he B ad- ag o w b ada idin. E ec o he C- e minal B ad- ag sequence o he old o b ada idin In w b ada idin, he open con o ma ion o he L3,4 loop accommoda es he amino acids o he B ad- ag sequence [42], whe eas in he bio in-complex s uc u e o co e-b ada idin he L3,4 loop adop s a closed con o ma ion (Fig 7). The mos d ama ic di e ences a e ound in he coo dina es o esidues Ala35-Glu41 o he L3,4 loop and he con o ma ion o he side chain o Ty 31 is also di e en in hese s uc u es. As men ioned abo e, he con o ma ion o Cys39, and he posi ion o i s Cαa om, a ies also be ween hese wo b ada idin s uc u es e en hough Cys69, which pai s wi h Cys39, has he same con o ma ion in bo h s uc u es. The impo ance o he equi alen disul ide b idge o bio in binding o shwana idin and hiza- idin has been ecen ly demons a ed using mu agenesis analysis [20]. This disul ide b idge has also been sugges ed o be impo an o bio in binding in hoe a idin and, in gene al, o all dime ic A ds [21], whe e he disul ide b idge is conside ed o main ain he L3,4-loop in he closed con o ma ion. In e es ingly, se e al di e en c ys al s uc u es o hoe a idin we e ecen ly de e mined by A aham e al. (2015), including in ac hoe a idin wi h i s C- e minal, Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 9 / 21 o he p o eins we e analyzed using SDS-PAGE (15%) in educing condi ions. In addi ion, he p o ein concen a ion was de e mined wi h a UV/Vis spec opho ome e (NanoD op 1000 Spec opho ome e , The mo Scien i ic, Wilming on, DE, USA) by measu ing he abso bance a 280 nm and using an ex inc ion coe icien o 43555 M –1 cm –1 and 43430 o co e-b ada idin V1 and CC mu an , acco dingly. Biophysical analysis o co e-b ada idin V1 & CC mu an The un olding empe a u e o co e-b ada idin V1 was analyzed using he VP-Capilla y DSC ins umen (GE Heal hca e, Mic oCal, No hamp on, MA, USA) in 50 mM sodium phospha e bu e (150 mM NaCl, pH 7.2) wi h p o ein concen a ion o 0.2 mg/ml. Solu ions we e degassed p io o measu emen s. Samples we e hea ed om 20˚C o 130˚C a a scanning a e o 2˚C/min. Feedback mode was se o ‘low’ and he il e pe iod was 5 s. The empe a u e ansi ion midpoin (T m ) was ob ained om he midpoin o he cu e ha was i ed o he da a a e i s sub ac ing he baseline om he measu emen da a and hen using he Le en- be g-Ma qua d non-linea leas -squa es me hod o i he cu e using he Mic oCal O igin 7.0 so wa e (Mic oCal, Mal e n Ins umen L d). Simila analysis was no possible wi h he CC mu an due o lack o p o ein o p ope analysis. The dissocia ion a e cons an (k diss ) o luo escen ly labelled bio in was de e mined by luo escence spec ome y using he bio in-labelled luo escen p obe A cDia™BF560 as desc ibed in [69]. In p ac ice, 50 nM dye in a bu e con aining 50 mM sodium phospha e, 650 mM NaCl and 0.1 mg/ml BSA (pH 7) was mixed wi h 100 nM co e-b ada idin V1 (o CC mu an ) and he change in luo escence in ensi y was measu ed o e ime. A 100- old mola excess o ee bio in (D-bio in, Sigma-Ald ich Co. LLC., S . Louis, MO, USA) was used o moni o he dissocia ion o his complex. The assay was pe o med a 50˚C using a Quan a- Mas e ™Spec o luo ome e (Pho on Technology In e na ional, Inc., Law ence ille, NJ, USA). Bio inyla ed BF560 was exci ed a 560 nm, and emission was measu ed a 578 nm. The a ini y o co e-b ada idin V1 owa ds B ad- ag (pep ide SEKLSNTK; GenSc ip , Pis- ca away, NJ, USA) was measu ed by ITC. The pu i ied co e-b ada idin V1 was dialyzed agains 50 mM sodium phospha e (pH 7.0) bu e con aining 100 mM NaCl, B ad- ag was dissol ed in he same bu e and he samples we e degassed using Mic oCal™The moVac. The analysis was pe o med a 40˚C using an iso he mal i a ion calo ime y VP-ITC Mic oCalo ime e (GE Heal hca e, Mic oCal, No hamp on, MA, USA) wi h 10 μl i a ion aliquo s o B ad- ag in 30 epea ed addi ions a in e als o 200 s using cons an s i ing speed o 440 pm. The da a we e analyzed wi h Mic ocal O igin 7.0 (Mic oCal LLC, No hamp on, MA, USA) so wa e. The obse ed eac ion hea s we e co ec ed by sub ac ing he hea o dilu ion caused by he i a- ion o he ligand alone in o bu e . K a ,ΔH and n (s oichiome y pe subuni ) we e ob ained h ough non-linea leas -squa es i o he co ec ed eac ion hea s o each i a ion s ep. Miscellaneous me hods PyMOL [60,61] and Bodil [62] we e used o analyzing s uc u es, isualiza ion and o c ea - ing igu es. The s uc u e-based sequence alignmen was done by Malign [67] o he Bodil so - wa e package o biomolecula isualiza ion and modeling [62]. A cu -o dis ance o 3.5 Å be ween non-hyd ogen a oms was used o hyd ogen bonds. Subuni one was used o c ea e all igu es unless no o he wise speci ied in he igu e legends. Suppo ing in o ma ion S1 Fig. Compa ison o he subuni IF1,2 in e ace esidues T p99, Leu91 and Ty 11 o co e-b ada idin (o ange; bold labels) [PDB:4BBO] and w b ada idin (magen a; labels in Co e-b ada idin PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0176086 Ap il 20, 2017 16 / 21 b acke s) [PDB:2Y32] based on supe imposi ion o he Cα aces o he p o eins. The bio- in molecule (BTN) o he co e-b ada idin s uc u e and he side chains o T p99, Leu91 and Ty 11 a e shown as s icks. Ni ogen a oms a e colou ed blue, oxygen a oms ed and sulphu a oms yellow. (TIF) S2 Fig. Compa ison o he e ia y s uc u e o w b ada idin [PDB:2Y32] o he homology models o hoda idin (Unip o : Q218I6; Genbank: WP_011472104.1) and b ada idin A2 (Genbank: WP_051334960.1). Supe imposi ion o he Cα aces o subuni I and subuni III o w b ada idin (magen a), hoda idin (blue) and b ada idin A2 (g ey) a e shown. The loop egions L1,2 o L7,8, and he N and C e mini, a e labelled. (TIF) S3 Fig. S uc u e-based sequence alignmen o co e-b ada idin, chicken A d, hiza idin, shwana idin, b ada idin II, hoe a idin, w b ada idin, hoda idin and b ada idin A2. The bio in-binding esidues ( op six s uc u es) a e ma ked wi h black squa ed boxes; he blue squa ed boxes indica e cys eine esidues o ming disulphide b idges in non- e ame ic A ds; he g een ‘1’ indica es he cys eine esidues o ming disulphide b idges in b ada idins and ho- da idin; he black iangle indica es he yp ophan esidue in equi alen posi ion o T p99 o he co e-b ada idin s uc u e ha is p esen only in e ame ic A ds; and he ‘B ad- ag’ esi- dues a e highligh ed wi h yellow backg ound. The be a-s ands 1–8 o co e-b ada idin a e labeled and indica ed by a ows. The conse ed esidues a e colou ed by he de aul scheme o he ESP ip 3 p og am (h p://esp ip .ibcp. /ESP ip /ESP ip /). (TIF) S1 Table. Yields o co e-b ada idin, co e-b ada idin V1 and CC mu an p oduced in E. coli BL21-AI. (DOCX) Acknowledgmen s We hank he bioin o ma ics in as uc u e suppo (J.V. Leh onen) om Biocen e Finland, and CSC IT Cen e o Science o labo a o y and compu a ional in as uc u e suppo . Docen Tiina Salminen is acknowledged o he excellen acili ies a he S uc u al Bioin o - ma ics Labo a o y and D . Heidi Kid on o he ini ial c ys alliza ion ials o co e-b ada idin. We acknowledge he MAXLAB, Sweden, o p o ision o synch o on adia ion acili ies, and we would like o hank he local con ac s o assis ance in using he beamline. We also acknowledge he in as uc u e suppo om Biocen e Finland o he Uni e si y o Tampe e P o ein Technologies co e acili y and aluable echnical suppo om Niklas Ka¨hko¨nen. We also acknowledge D . Tiina Riihima¨ki and D . Jenni Leppiniemi o hei aluable help in p o- ein exp ession and analysis. Finally, acknowledgmen s o D . Hen i No dlund, who had a i al ole o his wo k in he beginning o his s udy bu , un o una ely, passed away in 2008. Au ho Con ibu ions Concep ualiza ion: NA MSJ MSK VPH TTA. Da a cu a ion: NA TTA. Fo mal analysis: NA TTA VPH JAEM. Funding acquisi ion: VPH MSJ MSK TTA. 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