Jou nal o Gene al Vi ology (2000), 81, 1495–1505. P in ed in G ea B i ain
...................................................................................................................................................................................................................................................................................
A mul iply subs i u ed G–H loop om oo -and-mou h disease
i us in complex wi h a neu alizing an ibody: a ole o
wa e molecules
Wendy F. Ochoa,1Susana G. Kalko,1Mau icio G. Ma eu,2Paula Gomes,3Da id And eu,3
Es eban Domingo,2Ignasi Fi a1and Nu ia Ve dague 1
1Ins i u o Biologı!a Molecula de Ba celona, Consejo Supe io de In es igaciones Cien ı! icas, Jo di-Gi ona 18-26, 08034 Ba celona, Spain
2Cen o de Biologı!a Molecula ‘Se e o Ochoah, Consejo Supe io de In es igaciones Cien ı! icas, Uni e sidad Au o
!noma de Mad id,
28049 Can oblanco, Mad id, Spain
3Depa amen de Quı!mica O ga
!nica, Uni e si a de Ba celona, 08028 Ba celona, Spain
The c ys al s uc u e o a 15 amino acid syn he ic pep ide, co esponding o he sequence o he
majo an igenic si e A (G–H loop o VP1) om a mul iple a ian o oo -and-mou h disease i us
(FMDV), has been de e mined a 2n3A
/
esolu ion. The a ian pep ide includes ou amino acid
subs i u ions in he loop ela i e o he p e iously s udied pep ide ep esen ing FMDV C-S8c1 and
co esponds o he loop o a na u al FMDV isola e o sub ype C1. The pep ide was complexed wi h
he Fab agmen o he neu alizing monoclonal an ibody 4C4. The pep ide adop s a compac old
wi h a nea ly cyclic con o ma ion and a disposi ion o he ecep o - ecogni ion mo i A g–Gly–Asp
ha is closely ela ed o he p e iously de e mined s uc u e o he i al loop, as pa o he i ion,
and o unsubs i u ed syn he ic pep ide an igen bound o neu alizing an ibodies. New s uc u al
indings include he obse a ion ha well-de ined sol en molecules appea o play a majo ole in
s abilizing he con o ma ion o he pep ide and i s in e ac ions wi h he an ibody. S uc u al esul s
a e suppo ed by molecula -dynamic simula ions. The mul iply subs i u ed pep ide de eloped
compensa o y mechanisms o bind he an ibody wi h a con o ma ion e y simila o ha o i s
unsubs i u ed coun e pa . One wa e molecule, which o s e ic easons could no occupy he same
posi ion in he unsubs i u ed an igen, es ablishes hyd ogen bonds wi h h ee pep ide amino acids.
The cons ancy o he s uc u e o an an igenic domain despi e mul iple amino acid subs i u ions has
implica ions o accine design.
In oduc ion
Foo -and-mou h disease i us (FMDV) is he causa i e
agen o he mos economically impo an disease o clo en-
hoo ed animals wo ldwide (Pe ei a, 1981; B own, 1994). The
i us belongs o he genus Aph ho i us o he amily Pico na-
i idae (Ruecke , 1996). Con ol o he disease has been based
on la ge-scale accina ions wi h whole- i us inac i a ed
accines, limi a ion o animal mo emen s and des uc ion o
he ds exposed o he i us ( he ‘s amping-ou ’ p ocedu e)
( e iews in Bach ach, 1968; B own, 1994; Domingo e al.,
1990; Timoney e al., 1992). The a ailable accines show
Au ho o co espondence: Nu ia Ve dague .
Fax j34 93 2045904. e-mail n mc i!cid.csic.es
gene ally good p o ec ion agains in ec ion wi h homologous
i us and wi h an igenically ela ed isola es. Di icul ies acing
he e adica ion o FMD include he an igenic di e si y o
FMDV in na u e, which has been e lec ed in he iden i ica ion
o se en se o ypes (A, O, C, SAT1, SAT2, SAT3 and Asia1),
65 sub ypes, un il sub yping was in e up ed (Pe ei a, 1977),
and mul i udes o an igenic a ian s ha o en co-ci cula e in a
gi en geog aphical a ea (Ma eu e al., 1988). An igenic
a ia ion imposes a pe iodic upda ing o accine s ains,
an igenic p ope ies o which mus ma ch hose o he
ci cula ing i uses. Fu he mo e, some FMD ou b eaks ha e
been aced o accine s ains o he i us (Beck & S ohmaie ,
1987). Fo hese easons, i would be highly desi able o
de elop e ec i e, syn he ic FMD accines.
Neu alizing an ibodies a e impo an de e minan s o
0001-6861 #2000 SGM BEJF
W. F. Ochoa and o he sW. F. Ochoa and o he s
Table 1. Amino acid sequences o si e A pep ide a ian s
Pep ides A15-C"-B escia, A15-C-S8c1 and A15-C-S30 ep esen
sequences o FMDV isola es belonging o sub ype C". MAb 4C4,
which was aised agains C"-B escia, eac s wi h all h ee pep ides as
shown by immunochemical and s uc u al analyses (Ma eu e al., 1990,
1992; Ve dague e al., 1995, 1997). Syn he ic pep ide A15-C-
S8c1 LV p esen s a dec ease in binding a ini y o mos si e A-speci ic
MAbs, including 4C4 (Ma eu e al., 1992). k, Less han 20% o he
binding obse ed wi h he o he pep ides (j) in a quan i a i e
enzyme immunodo assay desc ibed by Ma eu e al. (1992). No i us
has been epo ed wi h a si e A sequence co esponding o pep ide
A15-C-S8c1 LV. The one-le e amino acid code is used.
Pep ide Sequence
Binding o
MAb 4C4
A15-C"-B escia YTASTRGDLAHLTATj
A15-C-S8c1 YTASARGDLAHLTTTj
A15-C-S8c1 LV YTASARGDLAHVTTTk
A15-C-S30 YTTSTRGDLAHVTATj
p o ec ion agains FMD and o he pico na i us diseases
(Ma eu, 1995; McCullough e al., 1992; Misbah e al., 1992).
An unde s anding o he ypes o in e ac ions be ween
an ibodies and i uses ha lead o i us neu aliza ion is
essen ial o accine design. One o he majo an igenic si es o
FMDV is loca ed in he G–H loop o capsid p o ein VP1 (Bi le
e al., 1982; P a e al., 1982; S ohmaie e al., 1982). This loop
is diso de ed on he su ace o FMDV pa icles (Acha ya e al.,
1989; Cu y e al., 1996; Lea e al., 1994, 1995; Logan e al.,
1993). Fo FMDV o se o ype C, his an igenic si e has been
e med si e A, and i beha es as an independen uni , wi h e y
limi ed in luence o o he capsid esidues ega ding he
in e ac ion o si e A wi h an ibodies (Hewa e al., 1997; Lea e
al., 1994; Ma eu, 1995; Ve dague e al., 1999). The beha iou
o his an igenic loop in FMDV pa icles in i s in e ac ion wi h
an ibodies can be mimicked ai h ully wi h syn he ic pep ides
ha ep esen he ele an amino acid sequences ound in
au hen ic i us (Cla ke e al., 1983; Ma eu e al., 1989,
1990; Ma eu, 1995; Rowlands e al., 1983). In e es ingly,
an igenic si e A includes a highly conse ed A g–Gly–Asp
(RGD) iple ha se es as he ecogni ion si e o an in eg in
ecep o (Be ins ein e al., 1995; Fox e al., 1989; He na
!ndez e
al., 1996; Jackson e al., 1997; Mason e al., 1994; Ne e al.,
1998).
The s uc u e o a syn he ic pep ide ep esen ing an igenic
si e A o FMDV C-S8c1 [a biological clone o na u al isola e C-
S a Pau Sp70 (Sob ino e al., 1983), a i us ep esen a i e o he
Eu opean sub ype C"FMDVs] in a complex wi h he Fab o
neu alizing monoclonal an ibodies (MAbs) aised agains he
i us has been s udied by X- ay c ys allog aphy (Ve dague e
al., 1995, 1996, 1998). The syn he ic pep ide spanned posi ions
136–150 o capsid p o ein VP1 (pep ide A15, Table 1). Two
complexes we e s udied, in ol ing MAbs SD6 (Ve dague e
al., 1995, 1996) and 4C4 (Ve dague e al., 1998). The wo
neu alizing MAbs we e aised agains wo di e en FMDV
ype C isola es (Ma eu e al., 1990): SD6 agains C-S8c1 and
4C4 agains C"-B escia, a i us ha di e s om C-S8c1 a wo
posi ions (140 and 149) wi hin si e A (Table 1). In he wo
complexes, he pep ide an igen acqui ed a e y simila quasi-
ci cula con o ma ion and he RGD mo i pa icipa ed di ec ly
in he in e ac ion wi h esidues o he complemen a i y-
de e mining egions (CDRs) o he an ibodies (Ve dague e al.,
1998). The RGD iple appea ed in an open u n con o ma ion,
e y simila o ha o educed FMDV pa icles o se o ype O
(Logan e al., 1993), and also simila o he con o ma ion in
RGD-con aining in eg in ligands (P a , 1997). Rema kably,
he Gly-142 and Asp-143 esidues o he RGD iple appea o
be c i ical o in e ac ion bo h wi h an in eg in ecep o and
wi h neu alizing an ibodies di ec ed o si e A (Ma eu e al.,
1996; Ve dague e al., 1995). Bo h s uc u al and biochemical
e idence sugges ha MAbs SD6 and 4C4 neu alize by
mono alen binding o an igenic si e A (Ve dague e al., 1997,
1998; e iew in Domingo e al., 1999).
The quasi-ci cula shape o he pep ide an igen in he
complexes was s abilized by pep ide–an ibody in e ac ions
and also h ough in apep ide hyd ogen bonds and an de
Waalshin e ac ions (Ve dague e al., 1995, 1996, 1998). I a
s uc u e simila o he one ound in hese complexes was also
main ained wi h a ian an igens (including one o se e al
amino acid subs i u ions), he s uc u e would gain g ound as
he basis o he design o new an i-FMD immunogens. Se e al
na u al isola es o FMDV o se o ype C ha e been cha ac-
e ized p e iously wi h ega d o he amino acid sequence a
an igenic si e A and eac i i y wi h MAbs (Ma ı!nez e al.,
1991; Ma eu e al., 1988, 1989, 1990; Ve dague e al., 1995,
1996, 1998; Villa e de e al., 1991). One o he isola es
analysed, e med C-S30 (o C"-Ba celona Sp 81), included ou
eplacemen s wi hin an igenic si e A. Two o he changes we e
coinciden wi h hose ound in he C"-B escia i us (Ala-140
Th , Th -149 Ala) and wo we e new (Ala-138 Th ,
Leu-147 Val) (Table 1). In spi e o he p esence o ou
amino acid subs i u ions in he G–H loop o VP1, he eac i i y
o FMDV C-S30 wi h MAb 4C4 was indis inguishable om
ha o FMDV C-S8c1 (Ma eu e al., 1990). Single subs i u ions
a posi ions 138 and, especially, 140 and 149 did no ha e a
signi ican e ec on he in e ac ion o syn he ic pep ide
an igens wi h mos MAbs, including 4C4, in ELISA (Ve dague
e al., 1998). Howe e , he single eplacemen o esidue Leu-
147, highly conse ed among FMDVs o se o ype C, by aline
diminished g ea ly he eac i i y o syn he ic pep ides wi h
mos MAbs, including 4C4, in enzyme immunodo assays and
compe i i e ELISA (Ma eu e al., 1992; Ve dague e al., 1998).
Tha is, he ou amino acid eplacemen s ound a he an igenic
si e A o C-S30 ela i e o C-S8c1 (Ma eu e al., 1989, 1990)
appea o exe a compensa o y e ec ha es o es he a ini y
o C-S30 (Ma eu e al., 1990) and o a syn he ic an igen
BEJG
S uc u e o a G–H loop a ian om FMDVS uc u e o a G–H loop a ian om FMDV
ep esen ing i s an igenic si e A (Ma eu e al., 1992) o MAb
4C4. The e o e, he subs i u ed an igen o e ed an oppo uni y
o in es iga e he s uc u e ha a a ian o m o an igenic si e
A could acqui e as a complex wi h an ibody. The esul s
desc ibed in his epo ea i m he quasi-ci cula con o ma ion
o his an igenic loop and e eal new ea u es abou he o ces
in ol ed in he s abili y o si e A and abou an igen–an ibody
in e ac ions, in pa icula , key in e ac ions media ed by wa e
molecules.
Me hods
MAb and syn he ic pep ides. The o igin and cha ac e iza ion o
he an i-FMDV MAb 4C4 (Ma eu e al., 1987, 1989, 1990) and he
p epa a ion and pu i ica ion o he Fab agmen o MAb 4C4 (Ve dague
e al., 1998) ha e been epo ed p e iously. Pep ides we e syn hesized by
solid-phase p ocedu es, pu i ied and analysed by using p ocedu es ha
ha e been desc ibed p e iously (Ca en
4oe al., 1992; Ma eu e al., 1996).
C ys alliza ion and da a collec ion. C ys als o he complex
be ween he Fab agmen o he MAb 4C4 and he a ian pep ide A15-
CS30 (Table 1) we e ob ained by he hanging d op- apou di usion
Table 2. Fea u es o c ys als o he an igen–4C4 Fab complex analysed in he p esen
s udy
Fea u e Value Re e ence alue Band wid h
C ys alliza ion and da a collec ion
Space g oup P2"2"2"
Cell pa ame e s (A
/)48n18, 69n32, 146n54
Resolu ion (A
/) 20–2n3
O e all comple eness (%) 93n0
A e age I σ7n5
Rsymm (%) 8n5
To al no. o esidues:
Fab 429
Pep ide 13
To al no. o sol en molecules 166
Volume o sol en (%) 49
Di ac ion ag eemen
Resolu ion (A
/) 15–2n3
Numbe o e lec ions 17943
R ee 0n262
R- ac o 0n248
RMSD om ideal dis ance:
Bond leng h (A
/)0n009
Bond angle (m)1n48
A e age he mal ac o (A
/#):
Fab 23n6
Pep ide 23n2
S e eochemis y o main chain:
Omega angle SD (m)1n26n03n0
Bad con ac s pe 100 esidues 0n87n610n0
Ze a angle SD1n33n11n6
S e eochemis y o side chain:
Chi-1 pooled SD 13n320n35n0
echnique and successi e mic o- and mac oseedings (S u a & Wilson,
1992). Typically small, wined needles, ob ained wi h 18% PEG 4K, we e
used o mic oseeding, which p oduced la ge needles a 16% PEG 4K.
Finally, hese la ge needles we e used o mac oseedings in 2 µl d ople s
con aining 7 mg ml Fab, 1n8mg ml pep ide, 6n5% PEG 4K, 0n2 M LiCl
wi h 50 mM T is–HCl (pH 9), equilib a ed agains a ese oi con aining
13% PEG 4K equally bu e ed a oom empe a u e. C ys als we e
o ho hombic, space g oup P2"2"2"wi h uni cell pa ame e s a l48n2A
/,
bl69n3A
/and c l146n5A
/, con aining one molecule o he complex
pe asymme ic uni , which co esponds o a sol en con en olume o
49%. A da a se was collec ed, a 100 K wi h 20% glyce ol as a
c yop o ec an , by using a Ma Reseach imaging pla e on a Rigaku
o a ing anode. In ensi ies we e e alua ed and scaled in e nally wi h
p og ams Denzo and Scalepack, espec i ely (O winowski & Mino ,
1996). Da a we e 93% comple e a 2n3A
/ esolu ion, wi h an in e nal
ag eemen ac o (Rsymm)o 8n5% (Table 2).
S uc u e solu ion and e inemen . C ys als o he complex
be ween pep ide A15-C-S8c1 LV and he Fab o MAb 4C4 seemed
ela ed o c ys als o med wi h he same Fab and pep ide A15-C-S8c1
( he names and amino acid sequence o pep ide an igens a e lis ed in
Table 1), whose s uc u e had been sol ed p e iously (Ve dague e al.,
1998). Howe e , he uni cell pa ame e c di e ed by abou 10 A
/and he
s uc u e was newly de e mined by molecula eplacemen by using he
BEJH
W. F. Ochoa and o he sW. F. Ochoa and o he s
Fig. 1. (A) S e eo iew o he (2Fo-Fc) elec on densi y map co esponding o he an igenic pep ide A15-C-S30. The pep ide
model and nea by sol en molecules (isola ed do s) a e also shown. (B) S e eo iew o he Fab–pep ide in e ac ions. Only he
Fab esidues in di ec con ac wi h he pep ide a e depic ed (open ods). Wa e molecules (W) in di ec con ac wi h he
pep ide o media ing he an ibody ecogni ion a e also shown. Hyd ogen bonds a e indica ed by b oken lines. (C) Supe -
posi ion o he A15 pep ide con o ma ions ound in he 4C4 Fab complexes wi h he A15-C-S30 ( illed) and he A15-C-
S8c1 (open) pep ides.
AMoRe package wi h he 4C4 Fab coo dina es as sea ching model
(Na aza, 1994). The ini ial solu ion was hen op imized by allowing o
mo e as ou sepa a ed igid bodies he a iable hea y, a iable ligh ,
cons an hea y and cons an ligh domains. The esul ing R- ac o was
0n36% in he esolu ion ange o 15 o 4 A
/. Examina ion o he elec on
densi y maps, calcula ed a his s age, clea ly showed ex a densi y
co esponding o he oligopep ide occupying he an igen-binding si e.
The inal model o he s uc u e o he complex was ob ained by i e a i e
cycles o model ebuilding by using he p og am O (Jones e al., 1991)
and posi ional e inemen wi h XPLOR (B u
$nge , 1992), including bulk
BEJI
S uc u e o a G–H loop a ian om FMDVS uc u e o a G–H loop a ian om FMDV
Table 3. Hyd ogen bonds be ween he A15-CS30 and A15-CS8 pep ides and he 4C4
Fab
The pa icipan s in he a ious hyd ogen bonds a e shown. C ys allog aphic da a a e desc ibed in Table 2 and
he h ee-dimensional s uc u es o he wo complexes a e shown in Fig. 2. The CDR loops a e gi en o
which he 4C4 Fab a oms lis ed belong.
Pep ide a om
Wa e
molecule 4C4 Fab a om CDR loop Dis ance (A
/
)
A15-CS30
Th -137 O – Asp-104 N H3 3n2
Th -137 Oγ1 – Se -103 OγH3 3n1
Th -138 Oγ1w8 – –2n7
Se -139 N w3 Se -95 O L3 3n0 2n7
Se -139 N w3 Asp-104 OδH3 3n0 2n8
Se -139 Oγ– Asn-96 Oδ1L3 2n7
Se -139 O w2 – – 2n7
Th -140 N w9 Se -31 OγL1 2n7 2n6
Th -140 Oγ1w9 – –3n2
Th -140 Oγ1w4 – –3n3
A g-141 O – Asp-98 N L3 2n9
A g-141 O w6 – – 3n0
A g-141 NH1 – Glu-97 Oε1L3 2n7
Gly-142 N w1 w5 Asp-98 Oδ1L32n9 3n1 2n8
Gly-142 N w2 – – 3n4
Gly-142 O w1 – – 2n9
Asp-143 N w2 – – 3n3
Asp-143 Oδ2 – A g-99 Nη2H3 3n0
Asp-143 Oδ1 – A g-99 NεH3 2n8
Asp-143 Oδ1 – Th -50 Oγ1H2 2n9
Leu-144 N w2 – – 2n8
Leu-144 O w8 – – 2n9
His-146 N – Ty -59 OηH2 3n4
His-146 Nδ1 – Ty -59 OηH2 3n1
His-146 Nε2 – Th -33 Oγ1H1 2n8
Val-147 O w8 – – 2n5
–w7 w1 – – 3n0
A15-CS8c1
Th -137 N – A g-54 Nη2L2 2n6
Th -137 O – Asp-104 N H3 3n2
Th -137 Oδ1 – Asp-34 Nδ2L1 2n9
Se -139 Oδ– Asn-96 Oδ1L3 3n4
Asp-143 O – Ty -59 OηH2 3n4
Asp-143 Oδ1 – A g-9 Nη2H3 2n7
Asp-143 Oδ2 – A g-99 Nη1H3 2n8
Asp-143 Oδ2 – Th -50 Oγ1H2 3n0
His-146 Nδ1 – Ty -59 OηH2 2n6
His-146 Nδ2 – Th -33 OδH1 3n2
His-146 Nδ2 – Se -52 OδH2 2n8
sol en co ec ion. The e ined model con e ged o c ys allog aphic
ag eemen ac o s R and R ee o 24% and 26%, espec i ely, o 17943
e lec ions in he esolu ion shell 15n0–2n3A
/(Table 2).
Molecula dynamics (MD) simula ion. Th ee se s o MD
simula ions, co esponding o he a iable module (F ) o he 4C4 Fab
in e ac ing wi h pep ides A15-C-S8c1, A15-C-S8c1 LV and A15-C-S30
(Table 1), we e pe o med by using he GROMOS96 package wi h i s
s anda d p o ein and wa e o ce ields ( an Guns e en e al., 1996).
C ys allog aphic coo dina es o pep ides A15-C-S8c1 and A15-C-S30
( esidues 137–148) we e used as s a ing models in he co esponding
simula ions. Th -148, no isible in he elec on densi y maps o he A15-
C-S8c1 complex, was added o a oid end e ec s in he c i ical Leu-147
esidue, by using he in o ma ion a ailable in he A15-C-S30 s uc u e.
S a ing coo dina es o he A15-C-S8c1 LV pep ide we e hen de i ed,
wi h he g aphic p og am TURBO (Rousel & Cambillau, 1989), by
BEJJ

W. F. Ochoa and o he sW. F. Ochoa and o he s
eplacing Leu-147 wi h aline. C ys allog aphic wa e molecules de-
e mined in he p esen wo k o he complex o 4C4 Fab wi h he A15-
C-S30 pep ide we e no included in he simula ions.
In e e y simula ion, he s uc u e o he co esponding complex was
placed ini ially a he cen e o a unca ed oc ahed on, he dimensions o
which we e chosen such ha he minimum dis ance o any p o ein a om
om he closes wall was 7 A
/. The edge leng hs o he co esponding
cubic boxes we e abou 73 A
/. Sys ems we e ea ed as imme sed in o an
equilib ium con igu a ion o bulk simple poin cha ge (SPC) wa e
(Be endsen e al., 1986). Wa e molecules ou side he box o wi h a
dis ance o a solu e a om o less han 2n3A
/we e emo ed. The numbe s
o wa e molecules conside ed in he h ee simula ions we e 5271, 5272
and 5264, espec i ely. To elax s ong wa e –wa e and wa e –p o ein
non-bonded in e ac ions, s eepes -descen ene gy minimiza ion was
pe o med un il s abiliza ion was eached. A e ha , coun e -ions we e
added o neu alize cha ged p o ein si es, wi h a subsequen ene gy
minimiza ion. Simula ions we e pe o med a cons an olume and
empe a u e (300 K) wi h pe iodic bounda y condi ions and an in-
eg a ion s ep o 2 s. Tempe a u e was kep cons an by weak coupling
o an ex e nal ba h (Be endsen e al., 1984, 1986). Bond leng hs we e
cons ained o equilib ium alues by using he SHAKE algo i hm
(Rickae e al., 1977). Equilib a ions we e achie ed wi hin 50 ps and,
a e wa ds, he h ee sys ems we e simula ed o a o al ime o 300 ps
wi h he o al po en ial ene gy emaining essen ially cons an . Only
amino acids om he pep ides and all wa e molecules we e allowed o
mo e du ing calcula ions. Analysis we e pe o med, mainly wi h
p og ams con ained in he GROMOS96 package, using he s uc u es
gene a ed e e y 0n1 ps du ing he in e al spanning om equilib a ion
un il he end ( om abou 50 o 350 ps).
Coo dina es. Coo dina es ha e been deposi ed in he B ookha en
p o ein da abase unde accession numbe 1EJO.
Resul s
S uc u e o he 4C4 Fab in complex wi h he
A15-CS30 pep ide
The inal elec on densi y maps o he complex be ween he
4C4 Fab and he A15-C-S30 pep ide, a 2n3A
/ esolu ion,
allowed he posi ioning o he main and side chains o 431 o
he o al o 436 4C4 Fab esidues (216 om he ligh chain and
220 om he hea y chain) and o 12 o he 15 pep ide esidues
(Fig. 1A). Hea y-chain Fab esidues 136–143, in he cons an
domain, and e minal pep ide esidues Ala-149, Th -150 and
he side chain o Ty -136 we e diso de ed, and hei co-
o dina es ha e no been in oduced in he p esen model. The
quali y o he inal maps is also e lec ed in he 166 well-de ined
sol en molecules ound, nine o which pa icipa e in he
in e ac ions be ween he pep ide and he Fab CDRs (Table 3),
and h ee wa e molecules es ablish b idges among pep ide
esidues (Fig. 1B).
The A15-C-S30 pep ide shows an o e all con o ma ion
closely ela ed o he one ound in o he si e A complexes
de e mined p e iously (Ve dague e al., 1995, 1998) (Fig. 1 C).
The RGD mo i , esidues 141–143, is loca ed in an open u n
con o ma ion p eceded by an ex ended egion, esidues
136–140, and ollowed by a sho helix, posi ions 144–148
(Fig. 1). All o he pep ide esidues inco po a ed in o he model
100
75
50
25
0
B
Residue numbe
Con ac a ea (% o esidue su ace)
100
75
50
25
0
A
136
Residue numbe
Con ac a ea (Å2)
137
*138
139
*140
141
142
143
144
145
146
*147
148
136
137
*138
139
*140
141
142
143
144
145
146
*147
148
Fig. 2. Con ac a eas o he A15 pep ides in he 4C4 Fab complexes wi h
he A15-C-S30 ( illed ba s) and he A15-C-S8c1 (open ba s) pep ides
exp essed as absolu e alues (A) o as pe cen ages o he esidue su ace
(B). As e isks indica e A15-C-S30 esidues ha di e in A15-C-S8c1.
Pep ide sequences a e shown in Table 1.
in e ac di ec ly wi h he hype a iable egions o he Fab
(Table 3 and Fig. 2). In pa icula , he en i e molecula su aces
o Asp-143, om he RGD mo i , and Leu-144 a e in con ac
wi h he an ibody, simila o wha had been obse ed in
complexes in ol ing Fab A15-C-S8c1 (Fig. 2) (Ve dague e al.,
1995, 1998). Howe e , he subs i u ed esidues Th -138 and,
pa icula ly, Th -140 and Val-147 do no p esen ex ensi e
in e ac ions wi h he an ibody (Fig. 2). The ou h subs i u ion,
Ala-149, no isible in he elec on densi y and no included in
he p esen model, mus also emain exposed o he sol en
and a om he an ibody.
Compa isons o he si e A a ian pep ides A15-CS30
and A15-CS8c1
The oo -mean-squa e de ia ion (RMSD) be ween he
main-chain pep ide a oms in he A15-C-S30 and A15-C-S8c1
Fab 4C4 complexes is only 0n4A
/ o he common esidues
137–147. The A g-141 side chain has di e en disposi ions in
he wo pep ides (Fig. 1C), al hough he elec on densi y
co esponding o his esidue was weak in bo h complexes
(Fig. 1A; see also Ve dague e al., 1995, 1998). The eplaced
esidues, Th -138 and Val-147, show he la ges main-chain
BFAA
S uc u e o a G–H loop a ian om FMDVS uc u e o a G–H loop a ian om FMDV
Fig. 3. Space- illing ep esen a ion o he h ee pep ide models A15-C-
S8c1 (A), A15-C-S8/LV (B) and A15-C-S30 (C). Pep ide esidues ha
con ibu e o in apep ide hyd ophobic in e ac ions a e highligh ed in da k-
blue. The wa e molecule ound in con ac wi h he subs i u ed Th -138 in
he A15-C-S30 pep ide is displayed in ed (C).
de ia ions o 0n7 and 1n0A
/, espec i ely. Despi e he high
s uc u al simila i y be ween he wo pep ides A15-C-S30 and
A15-C-S8c1, di e ences in he main-chain con o ma ional
angles a e impo an , pa icula ly a ound he RGD mo i (Fig.
2). Thus, in he A15-C-S30 pep ide, esidues Th -140 and A g-
141 a e si ua ed in he Ramachand an egion co esponding o
α-helices while, in he A15-C-S8c1 pep ide, Ala-140 and A g-
141 a e in he egions o β-s ands and le -handed helices,
espec i ely. A hi d si ua ion is ound in he s uc u e epo ed
o he G–H loop o he educed FMDV se o ype O"(Lea e al.,
1993, 1995), whe e he co esponding esidues Leu-144 and
A g-145 a e bo h ound in he Ramachand an egion co -
esponding o β-s ands. The lexibili y o Gly-142 allows
compensa o y main-chain o sional angles ha esul in he
o e all s uc u al simila i y o he h ee pep ides. I is impo an
o no ice he p esence, in he A15-CS30 s uc u e, o one wa e
molecule (named w8 in Fig. 1) ha is hyd ogen-bonded wi h
he side chain o Th -138 and he main-chain oxygen a oms o
esidues Leu-144 and Leu-147. This wa e molecule canno be
p esen , o s e ic easons, in he A15-C-S8c1 s uc u e, whe e
he bulkie Leu-147 side chain ills he a ailable space (Fig. 3).
MD analysis
Th ee MD simula ions co esponding o complexes o he
F 4C4 wi h pep ides A15-C-S8c1, A15-C-S8c1 LV and A15-
C-S30 we e ca ied ou as desc ibed in Me hods. The main
di e ence be ween he a e aged con o ma ions o he h ee
pep ides is he opening o he C- e minal egion in he
simula ion o he single-subs i u ed A15-C-S8c1 LV pep ide
(Fig. 4A). O he signi ican di e ences can also be seen a he
amino e mini and o he A g-141 side chain. The s abili y o
he a e aged con o ma ions was analysed by e alua ing he
RMSD alues o he pep ide luc ua ions in e e y simula ion.
The lowes alues co espond o he A15-C-S30 pep ide, while
he mo e cons an main-chain egion includes he RGD mo i
and Leu-144 in he h ee pep ides, wi h Asp-143 ha ing he
smalles a ia ion. The side chains o Asp-143 and Leu-144
also appea ex emely in a ian , p obably e lec ing he ex en
and he s abili y o he in e ac ions o hese esidues wi h he
an ibody. The s abili y o he e a-subs i u ed pep ide A15-C-
S30 obse ed du ing he simula ion is con ibu ed by a
hyd ogen-bonded wa e molecule loca ed in a si e e y close
o he posi ion o he b idging c ys allog aphic wa e (w8)
(Figs 1B and 4B). This wa e si e p esen s a high occupa ion
and a low RMSD o abou 0n1A
/. Hyd ogen-bond in e ac ions
ake place be ween he wa e molecule ha occupies his si e
and pola a oms om he subs i u ed esidues Th -138, Leu-
144 and Val-147 (Table 3). The inc eased hyd ophobic su ace
exposed o he sol en appea s o explain he depa u e om
he ‘co ec hsi e A-like con o ma ion obse ed by MD wi h
he A15-CS8 LV pep ide (Fig. 3).
BFAB
W. F. Ochoa and o he sW. F. Ochoa and o he s
Fig. 4. (A) S e eo iew o he supe posi ion o he A15-C-S8c1 ( illed), A15-C-S8/LV (shaded) and A15-C-S30 (open)
pep ides co esponding o he a e aged con o ma ions calcula ed in 300 ps o MD simula ions o he co esponding
F –pep ide complexes. (B) S e eo iew o he supe posi ion o he A15-C-S30 coo dina es om he c ys al ( illed) and he MD
simula ion (open). The wa e molecule ound in he c ys al s uc u e (shaded) is si ua ed close o he wa e si e de e mined in
he MD simula ion (open).
Discussion
The e ec s o single o mul iple amino acid subs i u ions a
an igenic si e A o FMDV o se o ype C on he eac i i y o
si e A-speci ic neu alizing MAbs ha e been s udied ex-
ensi ely by immunochemical assays employing a ian
i uses and subs i u ed syn he ic pep ides (Ma eu e al., 1989,
1990, 1992; Ma ı!nez e al., 1997; No ella e al., 1993;
Ve dague e al., 1998). Replacemen s a posi ion 147 al e ed
he binding o mos an ibodies subs an ially, including 4C4,
e en hough esidue 147 had only ma ginal in e ac ions wi h
ha an ibody. In pa icula , subs i u ion o Leu-147 by aline
diminished he binding o mos si e A-speci ic MAbs analysed
(Ma eu e al., 1992). Howe e , he e ec o his single
subs i u ion Leu-147 Val was appa en ly compensa ed o ,
a leas in pa , by he eplacemen Ala-138 Th (Ma eu e
al., 1992). These obse a ions and he ac ha na u al FMDV
isola e C-S30 (Ma ı!nez e al., 1991; Villa e de e al., 1991)
con ains hese c i ical eplacemen s in i s VP1 G–H loop
ela i e o clone C-S8c1 (Table 1) encou aged he p esen
s udy.
In he s uc u e o he 4C4 Fab complexed wi h he a ian
pep ide A15-C-S30 epo ed he e, he wo amino acids Th -
138 and Val-147 ha we e eplaced in he pep ide show only
mino in e ac ions wi h he Fab (Table 2 and Fig. 2). Di e ences
in binding a ini ies o pep ides wi h subs i u ions in hose
wo esidues should he e o e be due mainly o ee-ene gy
di e ences when adop ing a ‘co ec hsi e A-like loop s uc u e,
he one ecognized by an ibodies o by he ecep o (Fig. 5).
The educed s abili y o ha con o ma ion in he pep ide wi h
he single subs i u ion Leu-147 Val would hen explain he
educed a ini y obse ed o he an ibody. Ins ead, he double-
subs i u ed pep ide could eco e binding a ini y by e-
s abilizing he ‘co ec hcon o ma ion wi h he addi ional wa e
molecule (w8) ha b idges he chain hyd oxyl g oup o he
eplaced Th -138 wi h he main-chain oxygen a oms o
esidues Leu-144 and Leu-147 (Table 3 and Fig. 3C). In he MD
simula ion o he A15-C-S30 a ian pep ide, a wa e molecule
wi h a low RMSD is placed in almos exac ly he same loca ion
as he one ound in he c ys al s uc u e (Fig. 4B). These
s uc u al esul s p o ide a a ionale o how a gene al change
in speci ici y o a la ge di e si y o an ibodies can be achie ed
by a des abiliza ion o he o iginal an igen s uc u e. In hose
cases, u he subs i u ions can es o e he a ini y o he
an ibodies by inc easing he s abili y o he o iginal an igen
con o ma ion, e en when none o he esidues subs i u ed
BFAC
S uc u e o a G–H loop a ian om FMDVS uc u e o a G–H loop a ian om FMDV
Fabc ys al + Pep idec ys al Fab – Pep idecomplex
Fabsolu ion + Pep idesolu ion
∆Gbinding
∆G o al
∆G olding
Fig. 5. A simpli ied he modynamic cycle o a Fab in e ac ing wi h an
an igenic pep ide can be desc ibed as a wo-s ep p ocess. In he i s s ep,
he pep ide adop s indi idually he s uc u e seen in he complex ha is
o med in he second s ep, so ha ∆G o al l∆G oldingj∆Gbinding, whe e
∆G olding e e s o he ee-ene gy di e ence be ween he pep ide s uc u e
in solu ion and ha ound in he complex ( he Fab eo ganiza ion, i
equi ed, can also be included in his e m) and ∆Gbinding e e s o he ee-
ene gy di e ence due o he o ma ion o he in e ac ions be ween he
pep ide and he Fab. A ‘lock-and-key’ ecogni ion mechanism, in which
s uc u es emain unchanged on complex o ma ion, co esponds o
∆G olding l0. Ins ead, ∆G olding 0 desc ibes an ‘induced- i ’ mechanism,
whe e he complex would only be s abilized by a a ou able ∆Gbinding
con ibu ion. Analysis o Fab 4C4 complexes wi h a numbe o A15
pep ides, bo h om he a ailable c ys al s uc u es and wi h MD
simula ions, sugges ha ∆Gbinding(A15-C-S30) $∆Gbinding(A15-C-S8/LV),
while he expe imen al da a on he ela i e s abili ies o hese complexes
(Ma eu e al., 1992) imply ha ∆G o al(A15-C-S30) ∆G o al(A15-C-
S8/LV), which would allow us o conclude ha ∆G olding(A15-C-S30) 
∆G olding(A15-C-S8/LV). The educed s abili y o he ‘co ec ’ si e A-like
con o ma ion o he A15-C-S8/LV pep ide would explain he lack o
a ini y o mos si e A MAbs o his a ian pep ide (see ex ).
pa icipa es in di ec an igen–an ibody in e ac ions (F ei e,
1999).
Recogni ion o con inuous epi opes, like he VP1 G–H
loop o FMDV s udied in his wo k, depends no only on he
amino acid sequence bu also on he spa ial con o ma ion o
he epi ope, whe e sol en molecules o en play a c i ical ole.
In e p e a ion o mechanisms o i us escape om an ibody
ecogni ion, essen ial in RNA i us e olu ion and accine
design, mus also ake in o conside a ion he e ec s o amino
acid eplacemen s in connec ion wi h he sol en ea ange-
men s imposed. In spi e o di icul ies in p edic ing he e ec s
o amino acid eplacemen s in an igen–an ibody ecogni ion,
he p esen esul s o FMDV encou age he design o quasi-
cyclic s uc u es ep oducing an igenic si e A. Da a o he
G–H loop om a numbe o di e en an ibodies, including he
mul iply subs i u ed pep ide epo ed he e, sugges a obus
and s ongly immunogenic s uc u e despi e he mobili y o
his an igenic si e on he i ion su ace (Pa y e al., 1990;
Logan e al., 1993). Cock ails o quasi-ci cula pep ides
ep esen ing he mos equen escape mu an s ound a si e A
should be candida es o inco po a ion in o accine o -
mula ions. Such o mula ions should include addi ional B-cell
and T-cell epi opes o ensu e a b oad immune esponse o
minimize he selec ion o escape mu an s and accine ailu es
(Domingo & Holland, 1992; Taboga e al., 1997).
We wish o hank E. B occhi o he gene ous supply o MAb 4C4 and
he con inuous help wi h ou esea ch. Wo k in Ba celona was unded by
DGICYT g an s PB95-0218 and PB97-0873. Wo k in Mad id was
suppo ed by g an s om DGES PM97-006-C02-01 and Fundacio
!n
Ramo
!n A eces. W.F.O. is a ecipien o a ellowship om he Minis e io
de Educacio
!n y Cul u a (Spain). P.G. hanks he Fundaçao C. Gulbenkian
o a PhD ellowship and he Uni e si y o Po o o empo a y lea e.
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BFAD