Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC.

ARTICLE S uc u al analysis o ligand-bound s a es o he Salmonella ype III sec e ion sys em ATPase In C I onne Be nal 1 | Jonas Röme mann 1 | La a Flach 1,2 | Michele Lunelli 1 | Cha lo e Ue ech 2,3 | Michael Kolbe 1,4 1 Depa men o S uc u al In ec ion Biology, Cen e o S uc u al Sys ems Biology (CSSB), Helmhol z-Cen e o In ec ion Resea ch (HZI), Hambu g, Ge many 2 Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g, Ge many 3 Eu opean XFEL GmbH, Schene eld, Ge many 4 MIN-Facul y Uni e si y Hambu g, Hambu g, Ge many Co espondence Michael Kolbe, Cen e o S uc u al Sys ems Biology, Helmhol z-Cen e o In ec ion Resea ch, Depa men o S uc u al In ec ion Biology, No kes aße 85, 22607 Hambu g, Ge many. Email: michael.kolbe@helmhol z-hzi.de Funding in o ma ion F ee and Hansea ic Ci y o Hambu g; Ge man Fede al Minis y o Heal h; Helmhol z Associa ion unding agency IVF (Ini ia i e and Ne wo king Fund); "P omo ion o young CSSB scien is s" p og am by he Joachim He z S i ung; Leibniz-Gemeinscha , G an /Awa d Numbe : SAW-2014-HPI-4; Eu opean Resea ch Council unde he Eu opean Communi y's Se en h F ameweo k P og amme Abs ac T ansloca ion o i ulence e ec o p o eins h ough he ype III sec e ion sys em (T3SS) is essen ial o he i ulence o many medically ele an G am-nega i e bac e ia. The T3SS ATPases a e conse ed componen s ha speci ically ecognize chape one–e ec o complexes and ene gize e ec o sec e ion h ough he sys em. I is hough ha unc ional T3SS ATPases assemble in o a cylind ical s uc u e main ained by hei N- e minal domains. Using size-exclusion ch oma og aphy coupled o mul i-angle ligh sca e ing and na i e mass spec ome y, we show ha in he absence o he N- e minal oligome iza ion domain he Salmonella T3SS ATPase In C can o m monome s and dime s in solu ion. We also p esen o he i s ime a 2.05 Å esolu ion c ys al s uc u e o In C lacking he oligome iza ion domain (In CΔ79) and map he amino acids sugges ed o ATPase in e subuni in e ac ion, binding o o he T3SS p o eins and chape one–e ec o ecogni ion. Fu he mo e, we alida e he In C ATP-binding si e by co-c ys alliza ion o In CΔ79 wi h ATPγS (2.65 Å) and ADP (2.80 Å). Upon ATP-analogue ecogni- ion, hese s uc u es e eal emodeling o he ATP-binding si e and con o ma ional changes o wo loops loca ed ou side o he ca aly ic si e. Bo h loops ace he cen- al po e o he p edic ed In C cylinde and a e essen ial o he unc ion o he T3SS ATPase. Ou esul s p esen a ine unc ional and s uc u al co ela ion o In C and p o ide u he de ails o he homo-oligome iza ion p ocess and ATP- dependen con o ma ional changes unde lying he T3SS ATPase ac i i y. KEYWORDS ATPase, bac e ial pa hogenesis, c ys allog aphy, mul i-angle ligh sca e ing, na i e mass spec ome y, Salmonella en e ica, spec oscopy, ype III sec e ion sys em (T3SS) Signi icance s a emen : Pa hogenic G am-nega i e bac e ia use a conse ed ATPase o aid he deli e y o i ulence ac o s ac oss he ype III sec e ion sys em (T3SS) du ing he in ec ion o human cells. Ou high- esolu ion s uc u es o he Salmonella ATPase, named In C, e eal how ATP-analogues migh induce allos e ic egula ion o i s ATPase ac i i y by mo ing wo loops in ol ed in he sec e ion o i ulence ac o s. These obse a ions shed no el s uc u al and unc ional insigh s in o he mechanism o ac i a ion o T3SS-associa ed ATPases. Recei ed: 5 June 2019 Re ised: 2 Augus 2019 Accep ed: 6 Augus 2019 DOI: 10.1002/p o.3704 This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed and is no used o comme cial pu poses. © 2019 The Au ho s. P o ein Science published by Wiley Pe iodicals, Inc. on behal o The P o ein Socie y. 1888 P o ein Science. 2019;28:1888–1901.wileyonlinelib a y.com/jou nal/p o 1|INTRODUCTION The injec isome ype III sec e ion sys em (T3SS) is a mul i- p o ein nanomachine essen ial o he i ulence o many pa hogenic G am-nega i e bac e ia, including Salmonella, Shigella,Ye sinia, en e opa hogenic Esche ichia coli,Chla- mydia, and Pseudomonas ae uginosa ha cause millions o dea hs wo ldwide each yea . 1–3 The T3SS o ms a sy inge-like s uc u e ex ending om he bac e ial cy osol ac oss he bac e ial memb anes o he a ge cell o di ec ly injec i ulence e ec o p o eins in o i s cy oplasm. Al hough he s uc u al componen s o he T3SSs a e highly conse ed among bac e ial species, he sec e ed e ec o s a e pa hogen-speci ic. 1,4–6 Mos o he e ec o p o eins equi e he o ma ion o complexes wi h hei T3SS chape ones p io sec e ion. The chape ones main ain a egion o he e ec o s pa ially un olded o acili a e hei subsequen sec e ion h ough he na ow ape u e o he T3SS channel (20 Å). 7–9 The sec e ion mechanism o he T3SS c i ically depends on he hie a chical selec ion and deli e y o e ec- o s by he so ing pla o m complex. 10 The Salmonella pa hogenici y island 1 (SPI-1) so ing pla o m is a dynamic complex ha in e ac s wi h he cy osolic in e ace o he memb ane-embedded T3SS and o ms cy osolic soluble in e media es. 11,12 This complex is cons i u ed by a cen al ATPase oligome ic cylinde o med by In C (Sc N in he uni ied nomencla u e), which is linked h ough i s nega i e egula o O gB (Sc L) o he lagella C- ing o hologue SpaO (Sc Q) and he accesso y p o ein O gA (Sc K). Addi- ionally, In C in e ac s h ough i s cen al po e o he s alk p o ein In I (Sc O). 13,14 In C has been p oposed o ecognize and dissemble he chape one–e ec o complexes and un old he e ec o s in an ATP-dependen manne . 7 This unc ion mi o s he AAA+ ATP-d i en anslocase mechanism, which consis s o hyd olyzing ATP o powe con o ma ional changes o i s homo-hexame ic cylinde a chi ec u e and o igge un olding and ansloca ion o subs a es h ough he cen al po e o he cylinde . 15 Howe e , he T3SS ATPases sha e high sequence homology and h ee-dimensional s uc u al simila i ies wi h he βsubuni o he F 1 F O ATPases. 16,17 Recen ly, he E. coli ATPase EscN has been shown o o m a homo-hexame ic cylinde wi h six ATP-binding si es loca ed a he in e ace o adjacen dime pai s, sugges ing coope a i i y be ween subuni s. 18 EscN binds o i s cen al s alk p o ein and p esen s di e en unc ional s a es suppo ing a o a y ca aly ic mechanism homologous o he F 1 F O ATPases. in i o and in i o s udies show ha he T3SS ATPases o m oligome s in solu ion and in associa ion wi h he T3SS. The s oichiome y o he complexes in solu- ion ange om dime s o dodecame s depending on he bac- e ial species. 16,19,20 The T3SS ATPases old in h ee domains. The N- e minal domain is conside ed o be impo - an o s able assembly o highe oligome s; i binds o he ATPase nega i e egula o (O gB in Salmonella) and p e- sen s lipid a ini y. 20–24 The p edic ed ATPase co e is he cen al and mos conse ed domain. I con ains he phospha e-binding loop (P-loop) wi h he Walke box A mo i (GxGKT/S) cha ac e is ic o enzymes wi h ATP ac i i y. 25 The C- e minal domain has mode a e sequence simila i y among di e en species and is he po en ial ecog- ni ion si e o chape one–e ec o complexes. 22 The a omic s uc u es o he Shigella Spa47, E. coli EscN, Salmonella SPI-2 SsaN, and Salmonella Flagella FliI ATPases sha e high simila i y in con o ma ion and ATP-analogues binding s a es. 16,17,26,27 Al hough he T3SS ATPases a e conse ed, li le is known abou he s uc u e o he Salmonella Typhimu ium SPI-1 ATPase In C. He e, we p esen he i s s uc u e o he Salmonella T3SS ATPase In C lacking he i s 79 esidues (Δ79) in he apo-s a e and in he p esence o ATP-analogues. We show ha Salmonella In CΔ79dime izesinsolu ionin he absence o i s N- e minal domain. Ou s uc u al assignmen s allow mapping o amino acids and u he in e p e a ion o p e ious gene ic and biochemical analysis undamen al o unde s anding he unc ion o T3SS ATPases. Addi ionally, we cha ac e ize he s uc u e o In CΔ79 in p esence o ADP o ATPγS. These s uc u es e eal addi ional con o - ma ional changes o wo loops ou side o he ATP ca aly ic si e ha ha e been shown o be essen ial in he o e all unc- ion o In C. These s uc u al e idences p o ide insigh s in o he ene gizing mechanism o T3SS ATPases. 2|RESULTS 2.1 |In CΔ79 o ms monome s and dime s in solu ion The Salmonella In C belongs o he conse ed amily o T3SS ATPases, sha ing be ween 38 and 57% sequence iden- i y wi h i s o hologues (Figu e S1). I was epo ed ha he N- e minal domain (amino acids 1–79) o he T3SS ATPases is in ol ed in memb ane ancho ing and homo-oligome s abiliza- ion. 21,22 When we ecombinan ly exp essed and pu i ied he ull leng h In C, he N- e minal domain su e ed as p o eoly- sis (da a no shown), sugges ing ha he N- e minal domain o he linke o his domain a e lexible egions. Hence, we emo ed he i s 79 amino acids o gene a e a cons uc con- aining he p edic ed ATPase and C- e minal domains ollowed by a S ep-Tag ha we e med In CΔ79. The E. coli T3SS ATPase o ms a homo-hexame ic cyl- inde wi h ATP-binding si es loca ed a he in e ace o adja- cen subuni s, 18 simila o he F 1 ATPases. We easoned ha in e ac ion be ween ATPase subuni s migh occu also in he absence o i s N- e minal egion and cha ac e ized he BERNAL ET AL.1889 molecula size o In CΔ79 in solu ion. Size-exclusion ch o- ma og aphy coupled o mul i-angle ligh sca e ing (SEC- MALS) analysis o In CΔ79 esul ed in wo elu ion peaks, a majo one assigned as Peak I and a mino one named Peak II. The weigh -a e aged molecula masses o Peaks I and II co esponded well o a monome (40 kDa) and a dime (80 kDa) o In CΔ79, espec i ely (Figu e 1a, S2). Ou esul s a e in line wi h he E. coli ATPase EscNΔ7 lacking he i s se en amino acids ha o ms dime s in solu ion. 16 Fu he analysis o elu ion Peak I by na i e mass spec ome- y (MS) con i med he p edominance o monome s in his ac ion e en hough some dime s we e also de ec ed. In con as , na i e MS o he Peak II p esen ed mos ly dime s and some monome s. The small amoun o ime s and e a- me s de ec ed a e likely unspeci ic clus e s inhe en o his me hod (Figu e 1b, Table S1). Toge he , hese esul s dem- ons a e ha In CΔ79 lacking he N- e minal domain exis s p edominan ly as monome in solu ion and can sel -associa e in o dime s. 2.2 |S uc u e o In CΔ79 To de e mine he s uc u e o In C, we pe o med c ys alliza- ion ials using he monome ic size-exclusion ch oma og aphy (SEC) ac ion o In CΔ79. We sol ed he X- ay c ys al s uc- u e o In CΔ79 in he absence o ATP-analogues a 2.05 Å esolu ion (Figu e 2, Table 1). The apo- o m olds in wo s uc- u al domains, he ATPase co e (amino acids R81 o T355) and he C- e minal domain (T356 o N431) simila o i s bac e ial o hologues. The ATPase ca aly ic co e is cons i u ed by he α/βRossmann old 25 wi h a pa allel nine-s anded wis ed β-shee lanked by h ee helices a one side, and ou helices a he o he one. The phospha e-binding loop mo i (P-loop) is cons i u ed by he amino acid sequence GCGKT (162–166) and is loca ed be ween α2andβ5 o he ATPase co e. The smalle C- e minal domain is composed o h ee helices and con ains a helix–loop–helix mo i ha is p oposed o in e ac wi h chape one and e ec o p o eins. 27,28 Analysis o he B- ac o alues o In CΔ79 a e e ine- men shows ha he ATPase co e has an a e age alue o 50.7. Mos o his domain is a he igid (blue) and con ains wo p o uding loops wi h highe mobili y (cyan o ed in Figu e 3a). The i s loop egion is loca ed be ween α5 and α6 (amino acids A255 o L282) and he second is be ween β9 and α7 (L304 o S320). The a e age B- ac o o he C- e minal domain is 75.3, showing a highe mobili y in com- pa ison wi h he ATPase co e. This domain con ains a hi d highly mobile loop egion be ween α10 and α11 (K368 o R400). The β9-α7 loop is highly conse ed among he T3SS ATPases and he o he wo loops show a mode a e sequence homology (Figu e S1). The h ee loops a e a anged in he same on o In CΔ79 o ming a mobile in e ace ha could po en ially be in ol ed in con o ma ional changes o in e ac- ion wi h o he molecules du ing he ATPase ac i i y. Taken oge he , In CΔ79 shows high h ee-dimensional s uc u al simila i y wi h o he T3SS ATPases including Spa47 (RMSD o 1.05 Å o 325 aligned Cα esidues), EscN (1.51 Å o 312 esidues) (Figu e S3), SsaN (1.63 Å o 293 esidues), and FliI (1.60 Å o 317 esidues). As p edic ed om he sequence alignmen (Figu e S1), he majo di e ences be ween T3SS ATPase s uc u es a e loca ed a he C- e minal domains, p obably due o i s sugges ed unc ion in ecognizing speci ic e ec o -bound chape ones. 7,27 2.3 |S uc u al mapping o unc ional amino acids Mu a ional analysis o In C and i s Shigella o hologue Spa47, iden i ied esidues R189 and R191 o he ATPase co e (ma ked in lilac in Figu e 2) as essen ial o ATPase ac i i y, homo-oligome iza ion, and ype III sec e ion. 22,29 In ou s uc u e, hese amino acids a e loca ed a he exposed su ace o In CΔ79 and hei side chains p esen high FIGURE 1 S oichiome y o In CΔ79. (a) SEC-MALS analysis o In CΔ79. The SEC p o ile (dRI, le axis) p esen s wo elu ion peaks, I and II. The weigh -a e aged mola masses (g ay, igh axis) ac oss he elu ion peaks co espond o monome ic (40 kDa) and dime ic (80 kDa) s a es o In CΔ79. (b) Rep esen a i e na i e MS analysis o SEC-peaks I and II demons a ing he monome ic (39,901.9 ± 0.6 Da) and dime ic s a e (79,808 ± 2 Da) o In CΔ79, espec i ely. Da k g ay bands highligh co esponding peaks om he wo spec a. Masses a e summa ized in Table S1 1890 BERNAL ET AL. mobili y as deno ed by hei B- ac o s (Figu e 3a). I can be concei ed ha R189 and R191 play a ole in dime iza ion o In CΔ79 in he absence o he N- e minal domain. The C- e minal domain o some T3SS ATPases con ains conse ed amino acids ha a e c ucial o ype III sec e ion. The In C amino acid Y385 was shown o be essen ial o sec e ion o la e e ec o s h ough he T3SS. The p e iously sugges ed mechanism included di ec in e ac ion o Y385 wi h e ec o –chape one complexes. 28 We show ha Y385 o ms a hyd ogen bond wi h he side chain o D394, keeping mos o i s su ace a ea bu ied wi hin he s uc u e (Figu e 3b inle ). Howe e , we canno disca d he possibili y o a s uc u al change a ound his amino acid upon ATP binding o chape one–e ec o in e ac ion. The amino acid E384 was epo ed o pa icipa e in he in e ac ion wi h he s alk p o ein in Esche ichia coli. 18 E384 is su ace exposed in ou s uc u e, and as Y385, is loca ed in he α10-α11 mobile loop (Figu e 3a). Addi ionally, he In C L376 was shown o play a ole in ecogni ion o chape one-bound o e ec o s. 7,27 Analysis o hyd ophobici y dis ibu ion o In CΔ79 shows ha L376 is pa o a hyd ophobic pa ch oge he wi h he amino acids L378, F379, I380, and L382 (Figu e 3b). These esidues, excep o L378, a e conse ed among T3SS ATPases and migh be impo an candida es o chape one–e ec o ecogni ion by nonpola in e ac ions. 2.4 |Con o ma ional changes associa ed wi h ATP-analogue binding o In CΔ79 in solu ion To unde s and he molecula mechanism o ATP ecogni ion o In C, we moni o ed he con o ma ional changes o In CΔ79 upon binding o di e en ATP-analogues by using Fou ie - ans o m in a ed (FTIR) and ci cula dich oism (CD) spec oscopy. ATPγS, AMP-PNP, o ADP sup- plemen ed wi h equimola concen a ions o magnesium ions we e used as ligands. FTIR di e ence spec oscopy o In CΔ79 wi h ATP-analogues showed a dec ease o abso - bance a 1655 cm −1 , indica ing a educ ion o α-helical con- en upon ligand binding (Figu e 4a). 30 The signal educ ion is mo e p onounced o he ADP- and AMP-PNP-bound o ms and mode a e o he ATPγS-bound o m. CD analysis o In CΔ79 indica es also a educ ion o he α-helical con- en (208 and 222 nm) in he p esence o ADP o AMP- PNP, while no majo in ensi y change was de ec ed o he ATPγS-bound o m (Figu e 4b). Taken oge he , hese esul s show ha In CΔ79 unde goes con o ma ional changes upon binding o ADP and AMP-PNP in solu ion, whe eas he s uc u al changes upon ATPγS in e ac ion a e ba ely de ec able. 2.5 |C ys al s uc u es o In CΔ79 in he p esence o ATP-analogues To u he analyze he con o ma ional changes o In CΔ79 upon ligand binding, we pe o med co-c ys alliza ion and soaking expe imen s o In CΔ79 wi h ADP, ATPγS, o AMP-PNP supplemen ed wi h magnesium ions. This allowed us o sol e he c ys al s uc u es o In CΔ79 co- c ys allized wi h ADP a 2.80 Å esolu ion and In CΔ79 bound o ATPγS by soaking expe imen s a 2.65 Å esolu- ion. Howe e , we could no assign any ligand densi y o In CΔ79 wi h AMP-PNP and magnesium ions. FIGURE 2 O e all a chi ec u e o In CΔ79. (a, b) Two iews o In CΔ79 p oduced by 90 o a ion showing seconda y s uc u e elemen s labeled as in Figu e S1. The P-loop egion is highligh ed in o ange and p e iously s udied amino acids a e p esen ed as s icks. R189 and R191 a e ela ed wi h in e subuni in e ac ion (lilac). 22 Y385 is essen ial o ull ype III sec e ion 28 and he conse ed E384 in e ac s wi h he s alk p o ein in EscN ( aspbe y). 18 L376 in e ac s wi h chape one–e ec o complexes (g een) 7 BERNAL ET AL.1891 In he In CΔ79 apo- o m, he pu a i e ligand-binding si e is occupied by se e al wa e molecules (Figu e 5). In bo h co-c ys al s uc u es, mos o he wa e molecules we e eplaced by he ligands. The modeling o ADP bound o In CΔ79 was challenging because o i s discon inuous den- si y, likely due o pa ial occupancy o he si e o lexibili y o he ligand. We could model he ligand be ween wo clea densi ies o he adenine and phospha e g oups. Howe e , no densi y o he ibose g oup was dis inguished (Figu e 5, S4). The phospha e g oups o he ADP molecule o m hyd ogen bonds wi h he In C P-loop amino acids G164 and T166. The adenine g oup in e ac s wi h a wa e molecule bound o V411 by hyd ogen bonds. In he ATPγS bound s uc u e, a magnesium ion is coo dina ed by he β- and γ-phospha es o he ligand, he side chains o D249 and T166, and one wa e molecule ha o ms hyd ogen bonds wi h he α-phospha e o ATPγS. The phospha e g oups o he ligand in e ac wi h he P-loop o In CΔ79 o ming TABLE 1 Da a collec ion and e inemen s a is ics In CΔ79 In CΔ79-ATPγS In CΔ79-ADP Da a collec ion Wa eleng h (Å) 1.0332 1.0332 1.0332 Space g oup P 6 5 P6 5 P6 5 Cell dimensions a, b, c (Å) 106.3, 106.3, 73.5 107.9, 107.9, 73.8 107.4, 107.4, 73.5 Resolu ion (Å) 100–2.05 100–2.65 100–2.80 (2.10–2.05) (2.71–2.65) (2.87–2.80) R me ge 0.102 (1.091) 0.100 (1.596) 0.086 (0.804) R meas 0.109 (1.167) 0.106 (1.694) 0.092 (0.864) CC 1/2 99.6 (66.0) 99.9 (54.1) 99.9 (87.5) I/σ(I) 11.35 (1.98) 16.58 (1.53) 13.17 (1.52) To al e lec ions 234,745 (16,821) 144,020 (9,272) 80,465 (5,394) Comple eness (%) 99.9 (99.2) 99.8 (97.5) 98.3 (95.5) Mul iplici y 7.9 (7.8) 10.0 (8.9) 6.8 (6.5) Re inemen Re lec ions used 29,752 14,382 11,813 R wo k /R ee 0.168/0.207 0.186/0.224 0.211/0.257 No. a oms P o ein 2,711 2,677 2,623 Ligands 8 57 86 Wa e 152 33 13 B- ac o s P o ein 56.35 78.85 94.55 Ligands 75.96 105.98 136.29 Wa e 56.00 71.17 83.29 R.M.S. de ia ions Bond leng hs (Å) 0.003 0.003 0.003 Bond angles () 0.615 0.663 0.644 Ramachand an alues Fa o ed (%) 97.14 93.88 92.88 Allowed (%) 2.86 5.25 6.23 Ou lie s (%) 0.00 0.87 0.89 Ro ame ou lie s (%) 0.69 6.64 6.43 Clashsco e 4.61 8.45 9.42 S a is ics o he highes - esolu ion shell a e shown in pa en heses. 1892 BERNAL ET AL. hyd ogen bonds wi h he amino acids G164 and T166, and a sal b idge wi h K165. The adenine g oup is s abilized by π-πs acking wi h Y338. The in e ac ion o ligands wi h In CΔ79 esembles he ATP-analogue binding obse ed o o hologue T3SS ATPases (Figu e 6). 16,17,22,31 Addi ionally, single mu a ions o G164 and K165 in he In C P-loop esul in loss o ATP-hyd olysis unc ion. 22 Ou s uc u es alida e he ele ance o his loop o ATP ecogni ion and allow us o u he analyze he p ope ies o he emaining In C ligand-binding si e. FIGURE 3 S uc u al analysis o In CΔ79. (a) S uc u e colo ed by B- ac o alues. Colo -coding ba shows lowe (blue) o highe ( ed) B- ac o alues o igid o mobile egions, espec i ely. Flexible loops and amino acids o in e subuni in e ac ion a e labeled as in Figu e 2. (b) S uc u e depic ed by 180 o a ion colo ed by highe ( ed) o lowe (whi e) hyd ophobici y o amino acids. The hyd ophobic pa ch con aining L376 is shown as inle . Y385 bu ied ( o ming hyd ogen bonds wi h D394) and E384 exposed in he loop a e also depic ed in he inle FIGURE 4 Con o ma ional changes o In CΔ79 upon ligand binding in solu ion. (a) FTIR di e ence spec a o In CΔ79 bound o ATPγS, ADP, and AMP-PNP in e e ence o i s apo- o m. (b) Backg ound-co ec ed CD spec a o In CΔ79 in he absence and p esence o ATP-analogues. A ows indica e changes o α-helical con en upon nucleo ide addi ion BERNAL ET AL.1893 2.6 |Remodeling o he ATP-binding si e upon ligand in e ac ion The phospha e g oups o he ATP-analogues in e ac wi h he In CΔ79 P-loop. Upon binding o ATPγS, K165 is displaced owa d he β-phospha e o he ligand in e ac ing by a sal b idge. The adenine g oup o he ATP-analogues binds o a hyd ophobic pocke o med by Y338, P410, V411, and M167 in he In C ligand-binding si e. This hyd ophobic pocke is in closed con o ma ion in he apo- o m and is opened upon in e ac ion wi h ATP-analogues (Figu es 5 and 6a,b). The side chain o M167 is o ien ed owa d V411 c ea ing an open ca - i y o s abilize he adenine g oup. In addi ion, he loop includ- ing V411 (α11-α12) is loca ed close o M167 su ounding and u he de ining he open hyd ophobic pocke (Figu e 5). A simila pocke is o med in he ligand-binding si e o o he T3SS ATPases, al hough he amino acid composi ion in his egion has li le sequence simila i y. 16,17,31 A compa ison wi h he nucleo ide-binding si e o he Shigella Spa47 and E. coli EscN T3SS ATPases shows ha he adenine double- ing om he ADP-In CΔ79 s uc u e is a he displaced om he inne egion o he hyd ophobic pocke bu s ill cap u ed on i s su - ace (Figu e 6). 2.7 |Ligand induced con o ma ional changes in wo luminal loops Analyzing he o e all s uc u es o he apo- and ligand bound s a es, he α-helical con en o In CΔ79 dec eased FIGURE 5 S uc u al changes o he In CΔ79 ATP-binding si e in he p esence o ATP-analogues. Analysis o ligand in e ac ion (le column) and elec on densi y maps wi h 2Fo-Fc con ou a 0.8–1.5σ( igh column). P-loop (G162 o T166) in e ac ing wi h he phospha e g oups is colo ed in ligh o ange and o he key amino acids o ligand ecogni ion a e colo ed in cyan. Con ac s in ol ed in ligand s abiliza ion a e indica ed by yellow dashed lines. ADP, ATPγS, and magnesium ion a e labeled 1894 BERNAL ET AL. modes ly upon ecogni ion o ADP. The ADP-In CΔ79 s uc u e p esen s loss o α-helical a angemen mos ly in he ex emes o α10 and α11 acing he α10-α11 loop. The ATPγS-In CΔ79 s uc u e p esen s a sca ce dec ease o α-helical con en in α10 (Figu es 7 and 8). The dec ease o he α-helical con en in he ADP-c ys al s uc u e and he pa ial main enance o he seconda y s uc u e in he ATPγS-In CΔ79 s uc u e ollow he same endency o he s uc u al changes de ec ed by FTIR and CD spec os- copy analysis. FIGURE 6 Opening o he hyd ophobic pocke a he In CΔ79 ATP-binding si e and compa ison wi h T3SS ATPase o hologues. Su ace and ibbon ep esen a ions colo ed by highe ( ed) o lowe (whi e) hyd ophobici y o amino acids showing (a) he Salmonella In CΔ79 apo- o m, (b) In CΔ79 in p esence o ADP (pu ple) and ATPγS (lime), (c) he Shigella Spa47Δ83 wi h ATPγS (PDB ID: 5ZT1) and (d) he E. coli EscNΔ102 wi h ADP (PDB ID: 2OBM). Ligands and side chains o amino acids s abilizing he ligands a e depic ed as s icks in he same o ien a ion as in Figu e 5 FIGURE 7 O e all s uc u al changes o In CΔ79 in he p esence o ADP. (a) 3D s uc u al alignmen o he apo- o m (blue) wi h he ADP bound o m (pu ple). Di e ences in loop con o ma ions a e highligh ed in ec angles and key amino acids colo ed as in Figu e 2a. The β9-α7 loop mo es a dis ance o app oxima ely 7.1 Å. (b) 90 o a ion iew showing ADP in e ac ion in he ligand- binding si e BERNAL ET AL.1895 The apo-, ADP- and ATPγS-In CΔ79 s uc u es showed con o ma ional di e ences o wo o he mos mobile loops (α10-α11 and β9-α7) (Figu es 7 and 8). The α10-α11 loop is loca ed a he C- e minal domain o In C and is pa o a helix–loop–helix mo i . No densi y was obse ed o his loop in co-c ys als while he p o ein backbone was be e de ined in he apo- o m, indica ing an inc ease o i s lexibili y upon ligand in e ac ion (Figu e S5). In ou s uc u es, α11 is closely ollowed by V411 o he ligand-binding si e. Upon ligand in e - ac ion V411 pulls α11 and ge s close o M167 (Figu e 5), pos- siblya ec ing heα-helical con o ma ion o α11, α10, and he espec i e loop. Some amino acids ele an o T3SS ATPase unc ion a e loca ed in he α10-α11 loop. Indeed, poin mu a- ions in he In C G383A, E384A, Y385A, and G388A we e shown o cause a dec ease o sec e ion o la e e ec o s by he T3SS. 28 Addi ionally, he amino acidL376 ha in e ac swi h chape one–e ec o p o eins 7 is also pa o his loop. The β9-α7 loop is loca ed nex o he P-loop and p esen s signi ican con o ma ional changes in he p esence o ATP-analogues. Upon binding o ADP and ATPγS, he β9-α7 loop swings away by app oxima ely 7.1 and 8.9 Å, espec i ely (Figu es 7 and 8, S5). Poin mu a ions in his egion o In C, including E306A, E308A, E309A, E310A, and D312A, we e epo ed o a ec sec e ion o la e e ec o s by he T3SS. 28 Simila ly, he co esponding β9-α7loopo heShigella Spa47Δ83 was shown o inc ease i s lexibili y upon in e ac ion wi h ATP-ana- logues. 31 In e es ingly, c yo-EM analysis o he E. coli EscN homo-hexame ic cylinde showed di e ences in he α10-α11 loop con o ma ion o each ligand-bound s a e and di e ences in he β9-α7 loop upon ligand ecogni ion. 18 To analyze he loca ion o he loops β9-α7andα10-α11 in a pu a i e hexame ic complex, we buil a model by supe pos- ing he In CΔ79 apo- o m o he EscN asymme ic cylinde (PDB ID: 6NJP) using Pymol (Figu e 9). 18 In he model, β9-α7 and α10-α11 a e loca ed a he inne po e o he cylinde whe e he s alk p o ein (In I in Salmonella) in e ac s wi h EscN. In addi ion, he hyd ophobic pa ch con aining he chape one–e ec o in e ac ing amino acid L376 7 is mos ly o ien ed o he inne po e bu a p ominen cle obse ed be ween wo subuni s could p obably allow he epo ed ec- ogni ion si e (Figu e 9a). Mo eo e , he P-loop, he amino acids pa icipa ing in homo-oligome iza ion o ATPase sub- uni s (R198, R191), 22,29 and he amino acid o s abiliza ion o ATP in o hologues (R349) 16 we e loca ed a he in e ace o he homo-hexame ic model (Figu e 9b). Ou esul s show ha In CΔ79 con ains wo loops ha unde go con o ma ional changes upon in e ac ion wi h ADP o ATPγS. The mo e- men o hese loops could be in ol ed in he ecogni ion o e ec o and chape one p o eins o in he in e ac ion wi h o he T3SS componen s including he s alk p o ein In I du - ing he dynamic p ocess o ype III sec e ion. 3|DISCUSSION The cy osolic in e ace o he memb ane-embedded T3SS seems o o m a he e o-complex wi h six o wel e ATPase subuni s. 11–14,32 Howe e , in he soluble s a e, he isola ed ull-leng h T3SS ATPases can o m ime s, hexame s, and dodecame s. 18,19,32 I was p oposed ha he N- e minal FIGURE 8 O e all s uc u al changes o In CΔ79 in he p esence o ATPγS. (a) 3D s uc u al alignmen o he apo- o m (blue) wi h he ATPγS bound o m (lime). Di e ences in loop con o ma ions a e highligh ed in ec angles and key amino acids colo ed as in Figu e 2a. The β9-α7 loop mo es a dis ance o app oxima ely 8.9 Å. (b) 90 o a ion iew showing ATPγS in e ac ion in he ligand- binding si e 1896 BERNAL ET AL.