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.